Objective: It is unknown whether interactions between HIV infection and the safety of botulinum toxin A (BTX) exist. Methods: We studied eight patients with HIV infection who were treated with BTX every three months for up to nine years. All patients were on antiretroviral treatment. The efficacy and safety of BTX were evaluated. Results: Indications for BTX treatment (including off-label use), dosage of BTX, and frequency of application did not differ as compared to non-HIV infected patients. BTX treatment was effective in all HIV infected patients during a long-term observation period without loss of efficacy and without clinically relevant side effects. Only one of the eight patients showed mild side effects due to BTX, and no clinical signs of antibody development were noted. We also observed no signs of interaction with antiretroviral treatment. CD4+ cell count and viral load remained stable during the observation period. Conclusions: We conclude that BTX treatment is safe and effective in the treatment of HIV infected patients who suffer also from a condition which can be treated by BTX. It is a therapeutic option in addition to oral medication for HIV infected patients. Botulinum toxin A (BTX) is a therapeutic option showing good efficacy in the treatment of different neurological symptoms. Main indications for BTX treatment comprise focal dystonia, spasticity, and—only for onabotulinum toxin B—chronic migraine ,2. BTX sClinically relevant movement disorders are identified in about 3% of all patients suffering from HIV infection ,5. FurthAs hospitals with both a specialized BTX clinic and a clinic for neurological manifestations of HIV infection, we were able to observe the treatment of HIV infected patients with BTX for long periods. In this paper, we report on the long-term safety and tolerability of BTX in HIV infected patients including data on the efficacy in these patients.We performed a retrospective chart analysis of all HIV infected patients of the Department of Neurology, University of Münster, and of the Department of Neurology, Krankenhaus Lindenbrunn, and selected those who were treated with BTX between 1996 and 2018 at least once. We registered demographic, clinical, laboratory, and neurological data and analysed the efficacy, safety, and tolerability of BTX treatment. The administration of BTX was performed according to the individual judgement of a physician experienced and board certified in BTX treatment . The dosage, sites, and timeline of BTX administration followed standard recommendations as provided in the scientific literature. The efficacy of BTX was judged by clinical examination and, in addition, if needed, by the extensor digitorum brevis test [A 42-year-old man complained about increased muscle tone in the right hand and forearm leading to problems of coordination about seven years after the diagnosis of HIV infection was made. He received cART including tenofovir, emtricitabine, and atazanavir. Laboratory analysis before BTX treatment showed 670 CD4+ cells/µL, and plasma viral load was <50 copies/mL. The HIV stage was CDC B2; as a neuromanifestation of the HIV infection, the patient suffered from a mononeuropathy of the right sciatic nerve independently from the dystonia. Family history, MRI of the brain including the cervical spine, and analysis of cerebrospinal fluid (CSF) were unremarkable. There was no history of neuroleptics. All diagnostic work-up did not reveal any underlying cause for this movement disorder. Therefore, idiopathic segmental dystonia was diagnosed. However, the induction of dystonia by the antiretroviral treatment cannot be excluded completely. Treatment with L-dopa, trihexyphenidyl, tetrabenazine, and baclofen was not successful over two years, and treatment with BTX was initiated. The patient received 200 mouse units (MU) of BTX in the flexor carpi radialis muscle and in the flexor carpi ulnaris muscle and did not exhibit any side effects. There was profound and constant alleviation of the segmental dystonia during an observation period of more than five years. However, fluctuations of the dystonia during the day course remained. CD4+ cell count (820/µL) and viral load (<50 copies/mL) remained stable during the observation period of nine years.A 39-year-old male patient had been diagnosed with HIV and infected with stage CDC C3 with a CD4+ cell count of 407/µL and a plasma viral load of 45,000 copies/mL. cART was started, consisting of nevirapine, zidovudine, and lamivudine. Five years later, the patient developed spastic tetraparesis that was more pronounced on the right side, in addition to gait disturbances. Dissociated sensory loss below Th10 dermatome was detected accompanied by urinary incontinence. MRI of the brain was unsuspicious, and MRI of the spinal cord showed increased signal hyperintensity from Th8 to Th10. Analysis of CSF resulted in normal cell count and elevated protein; viral load in CSF was <50 copies/mL. The diagnosis of HIV associated vacuolar myelopathy was made. Treatment with baclofen in a dose of 75 mg/d was without improvement over six months. Treatment with BTX  in the left ischiocrural muscles and in the adductor muscles, in the gastrocnemius muscle, in the flexor digitorum longus muscle, in the soleus muscle, in the tibialis posterior muscle, and in the flexor digitorum profundus muscle on both sides was started. Injections were continued every three months for up to six years. At the beginning of the treatment, the modified Ashworth scale (MAS) score was 4. During the last two years, dosage was augmented to 500 MU. BTX achieved good results in alleviating spasticity without any side effects, and the MAS score was 2. No interactions with cART could be detected. cART consisted of nevirapine, emitricitabine, and tenofovir. The CD4+ cell count was stable at 655/µL, with a recent plasma viral load of <50 copies/mL.A 54-year-old male patient was referred with cognitive disturbances and finally diagnosed as an HIV infected late presenter in CDC stage C3. The CD4+ cell count was 56/µL, and the plasma viral load was 45,660 copies/mL. A cART, including raltegravir, stavudine, and nevirapine was started, inducing good immunological recovery. Three years later, the patient decided to stop cART, and nine months later, left sided hemiparesis developed. The CD4+ cell count was 37/µL and the plasma viral load was 34,760 copies/mL. Progressive multifocal leukencephalopathy (PML) of the brain was diagnosed by CSF analysis detecting JC virus and typical lesions in MRI (in particular cerebellar lesions). cART including lamivudine, tenofovir, and efavirenz was started, which stopped progression of PML. Two years later, progressive cerebellar tremor developed with severe problems during all activities of daily life. Treatment using primidone and quetiapine was without any effect. Treatment with BTX of the right biceps brachii muscle, brachioradialis muscle, flexor carpi radialis muscle, flexor carpi ulnaris muscle and tibialis posterior muscle in a total dosage of 130 MU was started. The therapy induced a good reduction of cerebellar tremor in amplitude and improved activities of daily living enormously. This therapy continued every three months over a period of four years. The side-effects of BTX did not emerge. No interactions with cART could be detected. cART consisted of stavudine, raltegravir, and lopinavir. The CD4+ cell count was stable, showing at least 345/µL, and the plasma viral load was <37 copies/mL.A 43-year-old man was hospitalised because of slowly progressive left-sided spastic hemiparesis. A diagnosis of HIV infection stage CDC C3 with a CD4+ cell count of 82/µL and a plasma viral load of 37,560 copies/mL was made. PML was diagnosed by CSF analysis detecting JC virus and typical MRI lesions. A cART including lopinavir, nevirapine, and zidovudine was started. In addition, cidofovir for treatment of PML was given. Treatment of spasticity and severe pain by baclofen was initiated. Already at a low dosage of 10 mg/d, severe side effects such as fatigue, dizziness, and headache occurred. Therefore, treatment of the upper extremity using BTX was started. A total dosage of 60 MU BTX in the left trapezius muscle and biceps muscle was applied. In the follow-up-examinations, a significant reduction of spasticity (from MAS score 3 to MAS score 1) and pain  was observed. BTX therapy has now been occurring every three months over five years. There were no side-effects of BTX. No interactions with cART could be detected. cART consists now of tenofovir, emtricitabine, and lopinavir. The CD4+ cell count is stable, showing at least 675 CD4+ cells/µL, and the plasma viral load was <37 copies/mL.A 52-year-old female patient had been diagnosed with HIV infection for about 10 years in CDC stage C2 when she presented with pain in the neck and shoulders. Physiotherapy and treatment with NSAIDs gave her only partial relief, and NSAIDs were poorly tolerated. The CD4+ cell count was 782/µL, and the plasma viral load was <37 copies/mL. Beside typical distal symmetric polyneuropathy both clinically and in neurophysiological testings, no other manifestations of HIV infection could be detected at the time of presenting. The patient felt healthy otherwise. She was on cART, including tenofovir, stavudine, and emtricitabine. Since the patient was highly impaired by the pain, treatment with BTX was started. The injection was made according the follow-the-pain concept, with 25 MU in the splenius capitis muscle and in the trapezius muscle on either side . BTX treatment decreased the average pain significantly after about two weeks from 7 to 3 on a numeric rating scale. After the second injection, the patient complained of weakness of the neck with mild dropping of the head for some weeks. The exact injection sites were re-evaluated but no change of total dosing was made due to the wishes of the patient. The observation period is ongoing with recurrent very mild head dropping as a side effect of BTX. No interactions with cART have been detected. CD4+ cell count is stable, showing 775 CD4+ cells/µL and a plasma viral load of <37 copies/mL.A 58-year-old female patient had been diagnosed with HIV infection about 10 years previously and was in CDC stage C3 when she presented with a spastic tretraparesis. This had started about two years ago and lead to the detection of PML. After cART was introduced, PML activity stopped and the spasticity was not progressive any longer. She had spasticity in her distal right arm and distal left leg. Physiotherapy gave her only partial relief, and tizanidine and lioresal were not tolerated. The CD4+ cell count at time of admission was 902/µL, and the plasma viral load was <50 copies/mL. The patient complained of cognitive slowing but felt healthy otherwise. She was on cART. Since the patient was highly impaired by the spasticity (MAS score 4), treatment with BTX was started in the flexor muscles of the right arm (200 MU) and the gastrocnemius and tibialis posterior muscle of the left leg (50 MU each). After the first injection, the patient reported improvement of spasticity but was not yet satisfied with the result (MAS score 3). We increased the dose to 300 MU in the arm and added the soleus muscle of the left leg with 50 MU, resulting in a total dose of 450 MU. We have been injecting this patient now for over four years and could not detect any interactions with cART. The MAS scores for both arm and leg were consistently 2.2) and showed both spasticity including Babinski’s sign and muscular atrophy and fasciculations. The electromyography confirmed positive sharp waves and denervation in all extremities. In addition, we could observe tongue fibrillation. A diagnosis of amyotrophic lateral sclerosis (ALS) was made, possibly induced by HIV infection, which has been described in the literature [A 48-year-old male patient with HIV infection for over 12 years due to intravenous drug abuse presented with slowly progressive tetraparesis. At the time of hospital admission, he was very weak BMI 16.7 kg/m kg/m2 anA 49-year-old male patient attended the outpatient clinic because of right-sided facial pain. He developed this pain some weeks after a typical zoster ophthalmicus. The pain was located around the eye and in the distribution of the second trigeminal nerve branch with a mean intensity of 6 out of 10 on a numeric rating scale. The zoster manifestation was the reason to perform a blood test for HIV infection, which was positive. The patient showed at that time a CD4+ cell count of 302/µL with a viral load of 1000 copies/mL but was otherwise healthy and had no other symptoms of HIV infection; he received cART for about six months at admission. The neurological examination was unremarkable. The patient was treated with pregabalin up to 150 mg bid which gave him about 50% relief. He did not tolerate higher doses of pregabalin and refused to take any antidepressants. He wished further pain treatment without oral drugs and was therefore offered BTX which has some evidence in purely trigeminal pain treatment but is off label . Under BThe application of BTX represents a very rare therapy in HIV infected patients although it is very effective for movement disorders, spasticity, and in other off-label indications. Our patient sample is the first case series reporting that BTX can be administered to adult patients with HIV infection easily for approved indications such as spasticity and dystonia . In our We performed a thorough diagnostic work-up including MRI and CSF analysis of every single patient to clarify possible associations between HIV infection and neuromanifestations for which they were treated with BTX. Our patients had developed both primary neuromanifestations of HIV infection  and secondary neuromanifestations . In two of eight cases (#3 and #4), we observed a link between opportunistic infection (PML) and neurological symptoms. In one patient (#2), there was a primary neuromanifestation of HIV infection (vacuolar myelopathy), in one patient (#1) the link remains unclear , and in one patient (#5) there is probably no relation between HIV infection and indication for BTX treatment. Treatment of spasticity in two patients and of dystonia in one patient was in accordance with the indications BTX is approved for. If spasticity is taken as an indication for BTX in general, we administered BTX outside its approved indications only in three of our eight patients .The patients were treated regularly in three month intervals with BTX. The HIV infected patients in our study reported the onset of improvement about six days after treatment onset, the maximum improvement was about six weeks after treatment onset, and a diminishing efficacy after about 10 weeks similar to reports of patients without HIV infection was noticed. Therefore, we assume a similar efficacy profile in both groups.According to our clinical impression, no neutralizing antibodies to BTX have been developed. Even after up to nine years of application, there was no therapeutic failure. Development of antibodies, although rare, represents an important limitation to the application of BTX in general. It might be that the immunosuppressive properties of cART induce a protection against the development of antibodies and that therefore no development of antibodies was observed.All patients but two (No. 4 and No. 7) received BTX treatment in a state of good immunocompetence. Viral loads were low or undetectable under cART therapy, CD4+ cell count was normal and kept stable over the time of treatment in six patients. As we have treated only two patients in a severe immunosuppressed state, we cannot be sure about safety and tolerability in immunocomprised patients.The results of this study are in accordance with reports on the application of BTX in four HIV infected children suffering from spasticity  and in aIn summary, BTX represents an effective treatment for spasticity and movement disorders in HIV infected patients with a reasonable safety-efficacy profile when conventional treatment  is not tolerated or ineffective. In addition, the off-label use of BTX in pain treatment also showed good results in two patients with HIV infection.


Zero risk of linked HIV transmission in serodiscordant couples when the HIV-infected partner had viral load less than 200 copies/ml (‘U status’) was found in observational studies. We aimed at estimating the proportion of time in which ‘U status’ was maintained and identifying factors associated with the risk of losing it.Observational cohort study.We included participants in the ICONA cohort who had reached an established ‘U status’  as of December 2010. The outcome was the number of person-days of follow-up (PDFU) above a viral load greater than 200 copies/ml, relative to the total number of PDFU observed. A logistic regression model was used to identify factors independently associated with the risk of losing ‘U status’.Eight thousand, two hundred and forty-one persons living with HIV were included in the analysis who contributed 2 670 888 PDFU. Of these, 1648 (20%) were women, 768 (9%) were people who inject drugs (PWID), and 2066 (25%) were foreign-born. The median of viral load measurements was 9 (IQR: 4–15). Overall, only 3.1% of PDFU were observed when viral load was above 200 copies/ml. The proportion of PDFU with viral load more than 200 copies/ml was higher than average in women (5.3%), unemployed (5.4%), PWID (4.7%), and in people with more than three previous virologic failures (6.3%). These variables were significant predictors of losing ‘U status’ in the multivariable logistic regression.Our results reinforce the validity of the U=U message in real-world setting. However, we identified subsets of our study population at higher risk of losing the ‘U status’ for whom additional efforts are needed. Since the end of the 1980s, several studies have investigated the association between HIV replication, as measured by plasma HIV-RNA or p24 antigen, and the risk of sexual transmission of HIV . In 1994After introducing combination antiretroviral therapy (cART), several investigators hypothesized that treatments could decrease the risk of sexual HIV transmission and that a person with an undetectable HIV-RNA may not transmit the virus .et al. suggested that there was no direct evidence that the HIV sexual transmission risk in serodiscordant couples was zero. Therefore, the definition of a transmission threshold was questionable or needed to be lower than previously believed  years, with a median of CD4The median of viral load measurements over the study period was nine IQR 4–15), and the median time with viral load above 200 copies/ml was 47.3 days (IQR 46.3–47.9). Thus, HIV-RNA was collected at least twice a year (average 2.5 times per year), which is the monitoring frequency by the cohort protocol. The main baseline patients’ characteristics have been summarized in Table 5, and thOverall, 617 participants (7.5%) spent less than 90% of PDFU with a viral load 200 copies/ml or less and were classified as losing their initial ‘U status’ over time. Approximately 65% of participants showed less than two values above 200 copies/ml over follow-up, and 30% of the participants experienced a single sustained episode, and approximately an additional 7% experienced two or more episodes .n = 617) with those who did (n = 7624), the first were more frequently female individuals (P < 0.001), PWID (P < 0.001), and foreign-born (P < 0.001). They were also less likely to have achieved a college or university degree (P < 0.001) and were more frequently unemployed (P < 0.001).At univariable analysis Table , when coP = 0.008), less likely having a co-infection (P < 0.001) and showed lower current and nadir CD4+ cell count (P < 0.001), higher peak median viral load (P < 0.001). Furthermore, they showed a longer time from HIV diagnosis to baseline (P = 0.002), had been followed up for longer, and were more likely to have previously experienced one or more episodes of virological failure (P < 0.001).Regarding HIV-related characteristics, PWH who had not retained the ‘U status’ overtime had also been more frequently previously diagnosed with AIDS . Of these, 96.9% of PDFU were spent with a viral load 200 copies/ml or less. Thus, only the remaining 3.1% of PDFU were observed when viral load was above 200 copies/ml. The highest proportion of time spent with a viral load above 200 copies/ml was observed in 2013, with a progressive decrease in the following years, especially after 2016 , foreign-born (5.4%), unemployed (5.4%), PWID (4.7%), and in people with more than three previous virological failures (6.3%). There was no evidence of a difference in proportions when comparing patients with ages between 18–45 and at least 46 years (Table The Prevention Access Campaign (PAC) launched the slogan ‘Undetectable=Untransmittable’ or ‘U=U’ in 2016. PAC officially started this U=U prevention campaign after the publication of two studies, HPTN052 and Partner 1 ,12. The However, in HPTN052 and Partner 1, the serodiscordant couples were both heterosexual and MSM. On the contrary, in Partner 2 and Opposites Attract, only MSM couples were included. In none of the four studies, data about PWID and foreign-born were available. Furthermore, information on whether participants have had an AIDS-defining disease or precedent virological failure were not recorded. Although there is no strong reason to think that results would have been different in different target populations, strictly speaking, the quantitative results seen in these studies do not apply to these different case-mix populations.Our analysis using the data of a cohort of PWH seen for care in Italy evaluated, for the first time, the probability of retaining an HIV-RNA 200 copies/ml or less (the ‘U status’) over a long period during the U=U prevention campaign era.We found that in our population of PWH, the ‘U status’ was maintained, on average, for 97% of the following 10 years of observation, and the proportion of PDFU spent in the ‘U status’ showed a trend for an increase in recent years. Although a causal link cannot be established and we cannot ascribe these results to the correct receipt of the U=U message because of very high rates of viral suppression, this data reassuringly suggests that U=U is an appropriate message to communicate to help decrease stigma and increase motivation to remain virally suppressed. Of note, although the risk of transmission was not evaluated in the analysis, our study population included MSM, heterosexual women, and men, as well as PWID and foreign-born, subpopulations, which were underrepresented in other studies.Furthermore, when fitting a multivariable logistic regression analysis, female sex, being PWID, foreign-born, and unemployed, were all independently associated with the risk of losing ‘U status’.Looking at each of these factors in detail, we found that women have lost the ‘U status’ more frequently than men (5.3 vs. 2.4%). The role of female sex as a risk factor associated with virological failure risk is debated in the literature . A meta-et al.[et al.[Foreign nationality has already been associated with an increased risk of virological failure. Saracino et al. in the sl.[et al. showed tl.[et al..et al.[The use of injection drugs was the most significant risk factor in losing ‘U status’ in our analysis. It has been previously associated with low adherence levels to cART, risk of treatment discontinuation, and virological failure. In the Swiss HIV cohort, Weber et al. conducteet al.. Furtheret al..et al.[et al.[PWH with poor education and those who were unemployed showed, in our analysis, a significant risk in losing the ‘U status’, compared, respectively, to PWH with advanced education and those employed. Many other studies demonstrated how these social determinants play a crucial role in response to antiretroviral therapy. D’Almeida et al. aimed thl.[et al. showed il.[et al.. Socioec+ cell count 200 cells/μl or less. In a cohort study with 5766 PWH, Armenia et al.[+ cell count as a predictor of virological failure is controversial. In the HPTN052 study, Eshleman et al.[+ cell count before starting cART have an increased virological failure risk. On the contrary, in their meta-analysis, Skowron et al.[+ cell count was a critical predictor of virologic suppression. More recently, Stirrup et al.[+ cell baseline is a predictor of virological failure with drug resistance in the UK CHIC study. Some of these discrepancies are likely to be because of the study design. For example, the CD4+ cell count measured at the time of viral suppression  is likely to be a predictor of subsequent risk of viral rebound as it reflects adherence to ART received before baseline.When considering virological and immunologic characteristics, losing ‘U status’ in our cohort was most frequently observed in PWH with a high peak viral load and in those with a CD4ia et al. demonstran et al. found thon et al. found thup et al. evidenceet al. showed how the risk of virological failures was decreasing in the last year; despite that, the risk associated with viral rebound according to the previous history of virological failure remained constant over time [et al.[Previous virological failures remained a significant predictor of losing ‘U status’ by multivariable model in our analysis. Moreover, PWH, who never experienced a virological failure, spent significantly lower PDFU with viral load above 200 copies/ml (2.9%) when compared with those who have had one to three (4.4%) and more than three previous episodes of virological failure (6.3%). These results were in accordance with previous observations from a number of other studies. Rusconi ver time . In the e [et al., who shoOur study has some limitations. Firstly, it can be argued that the definition of losing the ‘U status’ based on 10% of PDFU spent with a viral load above 200 copies/ml is arbitrary and highly dependent on the duration of follow-up. Indeed the longer the follow-up, the greater the chance to reach this endpoint. However, when we used the alternative endpoint of 5% of PDFU and of more than 3 months with a viral load greater than 200 copies/ml, the results were similar.Secondly, the frequency of viral load measurements is likely to vary from patient to patient, which could have affected the calculation of the PDFU. Specifically, PDFU could be more accurate for people with more frequent measures, although as we used a ‘most recent value carried forward’ approach, we believe that any bias is probably negligible. Also, we performed a sensitivity analysis in a subset of the cohort of participants whose HIV-RNA was monitored at least twice/year, and the results were again similar. If anything, the estimate of 3.1% of PDFU spent in the ‘U status’ is an under-estimate of what happened to the average person who is stably in care in our clinics.Thirdly, considering that our study is a retrospective analysis and no data about HIV-negative partners was available, we could not assess whether HIV sexual transmissions in PWH who have lost the ‘U status’ have occurred. Similarly, we identified factors associated with losing the ‘U status’, not the risk of HIV sexual transmission, and we are assuming that by reducing the proportion of people leaving the ‘U status’, we could achieve less new infections.Last but not least, adherence is a key unmeasured confounder in all these analyses, and failing to control for adherence could have biased the magnitude and significance of some of the associations described.With evidence coming from both clinical trials and observational studies supporting these efforts, clinicians should correctly communicate the U=U prevention message to the PWH –14. HoweIn conclusion, our data show an ecological correlation between the introduction of the U=U campaign and a scenario of unfrequent episodes of viral load greater than 200 copies/ml over a long period of time with a further trend toward a decrease after the launch of the campaign. U=U is an essential but straightforward campaign founded on scientific evidence. It has already successfully influenced public opinion, reducing the stigma in PWH and consequently improving their quality of life.Nevertheless, our and previous studies also show that more efforts need to be made to encourage clinicians to introduce this message during routine visits. Our data are useful to identify subpopulations (e.g. women and foreign-born) who may particularly benefit from targeted interventions.Icona Foundation Study Group: BOARD OF DIRECTORS: A. d’Arminio Monforte (President), A. Antinori (Vice-President), M. Andreoni, A. Castagna, F. Castelli, R. Cauda, G. Di Perri, M. Galli, R. Iardino, G. Ippolito, A. Lazzarin, G.C. Marchetti, G. Rezza, F. von Schloesser, P. Viale.SCIENTIFIC SECRETARY: A. d’Arminio Monforte, A. Antinori, A. Castagna, F. Ceccherini-Silberstein, A. Cozzi-Lepri, E. Girardi, A. Gori, S. Lo Caputo, F. Maggiolo, C. Mussini, M. Puoti, C.F. Perno. STEERING COMMITTEE: A. Antinori, F. Bai, A. Bandera, S. Bonora, M. Borderi, A. Calcagno, M.R. Capobianchi, A. Castagna, F. Ceccherini-Silberstein, S. Cicalini, A. Cingolani, P. Cinque, A. Cozzi-Lepri, A. d’Arminio Monforte, A. Di Biagio, R. Gagliardini, E. Girardi, N. Gianotti, A. Gori, G. Guaraldi, G. Lapadula, M. Lichtner, A. Lai, S. Lo Caputo, G. Madeddu, F. Maggiolo, G. Marchetti, C. Mussini, S. Nozza, C.F. Perno, S. Piconi, C. Pinnetti, M. Puoti, E. Quiros Roldan, R. Rossotti, S. Rusconi, M.M. Santoro, A. Saracino, L. Sarmati, V. Spagnuolo, V. Svicher, L. Taramasso. STATISTICAL AND MONITORING TEAM: A. Cozzi-Lepri, I. Fanti, P. Lorenzini, A. Rodano’, M. Macchia, A. Tavelli. COMMUNITY ADVISORY BOARD: A. Bove, A. Camposeragna, M. Errico, M. Manfredini, A. Perziano, V. Calvino. BIOLOGICAL BANK INMI: F. Carletti, S. Carrara, A. Di Caro, S. Graziano, F. Petroni, G. Prota, S. Truffa. PARTICIPATING PHYSICIANS AND CENTERS: Italy -- A. Giacometti, A. Costantini, V. Barocci (Ancona); G. Angarano, L. Monno, E. Milano (Bari); F. Maggiolo, C. Suardi (Bergamo); P. Viale, V. Donati, G. Verucchi (Bologna); F. Castelnuovo, C. Minardi, E. Quiros Roldan (Brescia); B. Menzaghi, C. Abeli (Busto Arsizio); L. Chessa, F. Pes (Cagliarti); B. Cacopardo, B. Celesia (Catania); J. Vecchiet, K. Falasca (Chieti); A. Pan, S. Lorenzotti (Cremona); L. Sighinolfi, D. Segala (Ferrara); P. Blanc, F. Vichi (Firenze); E. Pontali, M. Bassetti, A. Alessandrini, N. Bobbio, G. Mazzarello (Genova); M. Lichtner, L. Fondaco, (Latina); S Piconi, C. Molteni (Lecco); A. Chiodera, P. Milini (Macerata); G. Nunnari, G. Pellicanò (Messina); A. d’Arminio Monforte, M. Galli, A. Lazzarin, G. Rizzardini, M. Puoti, A. Castagna, F Gelpi, M.C. Moioli, R. Piolini, D. Bernacchia, A. Poli, C. Tincati, (Milano); C. Mussini, C. Puzzolante (Modena); P Bonfanti, G. Lapadula (Monza); V. Sangiovanni, G Di Filippo, V. Esposito, G. Di Flumeri, I. Gentile, V. Rizzo (Napoli); A.M. Cattelan, S. Marinello (Padova); A. Cascio, M. Trizzino ; D. Francisci, E. Schiaroli (Perugia); G. Parruti, F. Sozio (Pescara); C. Lazzaretti, R. Corsini (Reggio Emilia); M. Andreoni, A. Antinori, R. Cauda, V. Vullo, V Mazzotta, S. Lamonica, M. Capozzi, A. Mondi, A. Cingolani, M. Rivano Capparuccia, G. Iaiani, A. Latini, G. Onnelli, M.M. Plazzi, G. De Girolamo, A. Vergori (Roma); M. Cecchetto, F. Viviani (Rovigo); G. Madeddu, A. De Vito(Sassari); M Fabbiani, F. Montagnani (Siena); A. Franco, R. Fontana Del Vecchio (Siracusa); C. Di Giuli (Terni); G Calleri, G. Di Perri, S. Bonora, G.C. Orofino, M. Sciandra (Torino); A. Londero (Udine); V. Manfrin, G. Battagin (Vicenza); G. Starnini, A. Ialungo (Viterbo).Authors’ contribution: G.M., A.D.V., A.C.L., A.D.M., A.A., and E.G. conceived the study. A.C.L. performed the statistical analyses. A.D.V., A.C., F.M., C.F.P., R.G., G.M., and A.S. contributed to the patients’ enrolment and data collection. G.M., A.D., A.C.L., A.D.M., A.A., and E.G. wrote the first version of the manuscript. All authors reviewed the manuscript, provided critical scientific revisions, and approved the final version of the manuscript.G.M. reports personal fees from Gilead Sciences, personal fees from Janssen, personal fees from Merck Sharp and Dohme, personal fees from ViiV, outside the submitted work; F.M. reports grants and personal fees from ViiV, grants and personal fees from Gilead, personal fees from MSD, grants and personal fees from Jannsen, outside the submitted work; R.G. reports personal fees from ViiV, personal fees from Merck, grants from Gilead, outside the submitted work; A.S. reports grants from GILEAD, outside the submitted work; A.D.M. reports grants, personal fees, and nonfinancial support from Gilead Sciences, personal fees from Janssen-Cilag, personal fees from Merck, personal fees and nonfinancial support from ViiV Healthcare, outside the submitted work; A.A. reports grants, personal fees, and nonfinancial support from Gilead Sciences, grants and personal fees from Janssen-Cilag, personal fees from Merck, grants, personal fees, and nonfinancial support from ViiV Healthcare, outside the submitted work; E.G. reports personal fees from ViiV, personal fees from Gilead, grants from Gilead, grants from Mylan, personal fees from Mylan, personal fees from Angelini, outside the submitted work; A.D.V., A.C.L., A.C., C.F.P., and G.M. have nothing to disclose.


NCT03205917). The primary endpoints were safety, tolerability and pharmacokinetics for both parts and antiviral activity among viremic adults living with HIV and not on ART for part 2 of the study. The secondary endpoints were changes in CD4+ T cell counts and development of HIV-1 sequence variations associated with PGDM1400, PGT121 and VRC07-523LS resistance in part 2. Intravenously administered PGDM1400 was safe and well-tolerated at doses up to 30 mg kg−1 and when given in combination with PGT121 and VRC07-523LS. A single intravenous infusion of 20 mg kg−1 of each of the three antibodies reduced plasma HIV RNA levels in viremic individuals by a maximum mean of 2.04 log10 copies per ml; however, viral rebound occurred in all participants within a median of 20 days after nadir. Rebound viruses demonstrated partial to complete resistance to PGDM1400 and PGT121 in vitro, whereas susceptibility to VRC07-523LS was preserved. Viral rebound occurred despite mean VRC07-523LS serum concentrations of 93 µg ml−1. The trial met the pre-specified endpoints. Our data suggest that future bNAb combinations likely need to achieve broad antiviral activity, while also maintaining high serum concentrations, to mediate viral control.HIV-1 therapy with single or dual broadly neutralizing antibodies (bNAbs) has shown viral escape, indicating that at least a triple bNAb therapy may be needed for robust suppression of viremia. We performed a two-part study consisting of a single-center, randomized, double-blind, dose-escalation, placebo-controlled first-in-human trial of the HIV-1 V2-glycan-specific antibody PGDM1400 alone or in combination with the V3-glycan-specific antibody PGT121 in 24 adults without HIV in part 1, as well as a multi-center, open-label trial of the combination of PGDM1400, PGT121 and the CD4-binding-site antibody VRC07-523LS in five viremic adults living with HIV not on antiretroviral therapy (ART) in part 2 ( A combination of three monoclonal antibodies transiently reduced viremia in people living with HIV-1 and not on antiretroviral therapy, but it did not prevent viral rebound. Further studies are needed to determine if this approach can be optimized. Rapid selection of neutralization-resistant viral variants resulting in therapeutic failure has been observed in all referenced studies, and it has become evident that bNAb monotherapy is insufficient for viral control due to the frequent presence of pre-existing escape mutations in the substantially diverse within-host HIV quasispecies. Combination of two bNAbs with complementary epitope specificities—the CD4-binding-site (CD4bs) antibody 3BNC117 and the V3-glycan antibody 10-1074—were able to suppress viral rebound in a subset of individuals for an extended period during ATI; in contrast, viral breakthrough was observed in individuals in the presence of baseline escape or when one of the antibodies fell below the therapeutic threshold, resulting in functional monotherapy8.HIV-1-specific bNAbs targeting multiple epitope regions of the HIV-1 envelope trimer (Env) have demonstrated the ability to robustly reduce plasma viremia in people living with HIV not on ART as well as to modestly delay viral rebound in individuals during an analytical antiretroviral treatment interruption (ATI)9, and the combination of the CD4bs antibody VRC07-523LS, the V3-glycan antibody PGT121 and the V2-apex antibody PGDM1400 has been identified to neutralize 99% of a panel of 374 cross-clade HIV-1 strains, of which 82% would be neutralized with at least two active antibodies (with 80% inhibitory concentration (IC80) of <10 µg ml−1)9. However, it is important to remember that such panels reflect single variants and not the complexity of within-host diversity found in natural infection.It has, therefore, been postulated that three bNAbs targeting different epitope regions would be necessary to overcome viral variants with potentially pre-existent escape mutations and provide sufficient control of the virus to prevent development of novel resistance. Complementary viral coverage resulting in extended breadth and potency has been modeled for multiple bNAb combinations50) of 0.003 µg ml−1, being ten- to 100-fold more potent than CD4bs antibodies such as VRC01 and 3BNC117 (ref. 10). Indeed, PGDM1400 provided highly potent antiviral activity in non-human primate simian–human immunodeficiency virus (SHIV) SF162P3 challenge studies12. Here, we evaluated the safety, tolerability and pharmacokinetics of PGDM1400 when given intravenously, alone or in combination with PGT121 and VRC07-523LS, in adults without HIV and determined the antiviral activity of all three bNAbs in viremic adults living with HIV not on ART.Although both VRC07-523LS and PGT121 have demonstrated robust antiviral activity in viremic people living with HIV, PGDM1400 has not been evaluated in humans thus far. This antibody was originally identified in donor 84 of the International AIDS Vaccine Initiative (IAVI) Protocol G cohort and is exceptionally broad and potent, covering 83% of a panel of 106 cross-clade pseudoviruses at a median 50% inhibitory concentration  in adults without HIV. Each participant in part 1 received either PGDM1400 or placebo  or PGDM1400+PGT121 or placebo , randomized at a ratio of 3:1 each  or a single infusion of 30 mg kg−1 of PGDM1400+PGT121  in viremic adults living with HIV not on ART  estimates for the groups without HIV were 20.77 days  when given alone and 17.4 days  when co-administered with PGT121, respectively  . In the group with HIV, the median  elimination t1/2 estimates were 11 days  for PGDM1400, 11.8 days  for PGT121 and 29.3 days  for VRC07-523LS, given that the latter bNAb is equipped with the half-life-extending LS variant13. Serum neutralizing activity for each of the three bNAbs was also measured using a combination of three pseudoviruses, each with respective neutralization susceptibility to one but not the other two bNAbs as follows: 6540.v4.c1 (PGDM1400), CH505TF.N334S.N160A.N280D.1 (PGT121) and CAP220.2.00_A8_5B (VRC07-523LS). bNAb concentrations measured by binding and neutralizing antibody assays generally displayed moderate to substantial concordance (ρc > 0.9)  and TZM-bl neutralization assays were used to individually measure PGDM1400, PGT121 and VRC07-523LS levels in serum  Fig. .Fig. 1PG−1 each , and the mean drop in VL was −2.04 log10 copies per ml at nadir. Viral rebound, as defined by a confirmed increase of ≥0.5 log10 copies per ml above nadir, occurred between 13 days and 70 days after nadir  or days 21–85 after bNAb infusion, with VL levels trending toward day 0 pre-infusion levels. The one participant in group 3B (participant 693–7312), who received PGDM1400 and PGT121 at 30 mg kg−1 each, showed a decrease in plasma VL of −2.16 log10 (day 7 difference from mean of screening and day 0) with a nadir at day 6 after infusion, before VL rebounded by day 30 (24 days after nadir). The VL, however, remained lower than the pre-infusion baseline  until ART was started on day 108.Baseline viral loads (VLs) at the day of bNAb infusion in five viremic participants with HIV not on ART, enrolled in group 3, varied from 2,770 to 163,130 copies per ml  Fig. . One indach Fig. , all othEnv sequences from participant isolates with the matched neutralization data to identify the mutations underlying the resistance patterns  of circulating viruses were tested for bNAb sensitivity in TZM-bl assay at baseline and during viral rebound. In total, we generated 43 sequences from baseline and 37 sequences from rebound viruses . All participants either showed resistance to PGDM1400 and PGT121 at baseline (participant 693–2215) or showed viral escape at rebound . We then analyzed 14 and is strongly associated with susceptibility15. Each participant showed different patterns for losing this glycan site through mutations at either residue 160 and/or 162 , and multiple resistance mutations were also found within individual participants (693–1969 and 693–7989). For participant 693–2215, both baseline and rebound viruses were resistant to PGDM1400, likely explained by the presence of glycine at the Env site 166, instead of the more common arginine at this site. G-166 is a known resistance-associated mutation for PGDM1400 (ref. 15) at a critical bNAb contact site14. For participant 693–7989, two rebound viruses that were resistant to PGDM1400 did not show any canonical resistance mutations, such as those identified as signatures in Bricault et al.15 and from previous mutagenesis studies compiled on the Los Alamos HIV Databse CATNAP (https://www.hiv.lanl.gov/content/index)16. These two viruses did have a V120I mutation, which could be a candidate mutation for resistance based on its structural proximity to PGDM1400-like bNAb contact sites14.Viral escape from PGDM1400 for participants 693–1969, 693–7312 and 693–7989 was mediated by the loss of the potential N-linked glycosylation site (PNGS) at residue 160, which is a key Env glycan contact for V2 apex bNAbs17 and a requisite for susceptibility to such bNAbs15. This occurred by different routes across participants, either gaining PNGS at 334 (participants 693–2215 and 693–1969) or not (participants 693–7312 and 693–7989). In the latter two participants, 2–3 different resistance mutations per participant were found in the rebound viruses. Two rebound viruses in participant 693–7989 escaped PGT121 not by losing N332 glycan but, instead, by the D325N mutation, which is also a resistance signature for PGT121 (ref. 15) and was found in clinical escape from monotherapy with PGT121 (ref. 18) and with 10–1074 (refs. 8). For two rebound viruses from participant 693–7989 (A7.1 and A11.1), none of the V3 loop resistance mutations was found; however, they did have mutations that shifted a hypervariable V1 PNGS two sites toward the N-terminus relative to other viruses from this participant, and V1 loop glycans have been shown to affect V3 glycan bNAb sensitivity20.The escape from PGT121 occurred for all participants predominantly due to the loss of the PNGS at 332, which is a key contact glycan for V3 glycan bNAbs4, is only a limited snapshot of the full diversity within each participant, our analyses are not powered to address the question of whether the bNAb-resistant rebound viruses were present at low frequency in baseline plasma, were mutated from baseline viruses and/or were activated from the latent reservoir.Analyses of the phylogenetic relationships between baseline and rebound viruses showed that bNAb escape was polyclonal Fig. . Because80 increase was only 1.5-fold  to the CD4bs antibody 3BNC117  evolution of viral resistance to VRC07-523LS neutralization and (2) decay of VRC07-523LS concentrations to sub-therapeutic levels. Serum ID80 titers capture both of these mechanisms as they are based on both VRC07-523LS serum concentrations and the neutralization sensitivity of viruses  developed or not . This result suggests that viral escape from VRC07-523LS occurs below a common threshold of serum neutralizing activity against contemporaneous viruses, which can be reached by either viruses developing neutralization resistance (participant 693–2215) or decay of bNAb concentrations (participant 693–7989), or by a combination of both (participant 693–1969). Although the low number of participants precluded robust statistical analyses, these data still suggest that maintaining ID80 titers above the threshold of ~1,000 could improve viral control.To further understand why viral rebound occurs without developing substantial VRC07-523LS neutralization resistance, we analyzed VRC07-523LS serum IDnot Fig. . The med3 or dual therapy4, and during VRC01 monotherapy1, viral rebound in viremic individuals was observed at similar serum ID80 titer ranges for the respective bNAb, as compared to VRC07-523LS in this study , as was observed for VRC07-523LS in this study . Despite some minor differences, the IC80 values for rebound viruses across these previous studies were fairly similar to VRC07-523LS in this study. Together, these results suggest that, unlike the development of complete neutralization resistance against V2 apex and V3 glycan-targeted bNAbs by individual mutations at the target epitope, viral escape from CD4bs bNAbs involves a combination of the evolution of partial CD4bs bNAb escape as well as the decay of serum bNAb concentrations.Comparison to other clinical studies testing CD4bs bNAbs in a post hoc analysis showed that similar mechanisms of viral escape are at play. During both 3BNC117 monotherapyudy Fig. . Further80 titers for rebound viruses for the triple bNAb combination studied here were not significantly different than the dual bNAb therapy used in a previous study by Bar-On et al.4; and although the triple combination rebound ID80 titers in our study were ~5-fold lower than the dual combination in Bar-On et al.4, they tended to be higher than in the single bNAb studies  and its half-life-optimized version CAP256V2LS. This bNAb class, although with limited breadth against clade B viruses9, has superior potency compared to other bNAb classes and is, therefore, a promising component of bNAb combinations. Indeed, PGDM1400 is also being explored in combination with VRC07-523LS and PGT121 or 10-1074 in participants without HIV in the ongoing HVTN130 study (NCT03928821) with the goal to develop passive immunization strategies for HIV prevention.In this study, we established the safety and tolerability of the PGDM1400 antibody when administered intravenously, alone or in combination with the bNAbs PGT121 and VRC07-523LS in participants with and without HIV. To our knowledge, this is the first report of a triple antibody combination in humans for the treatment of HIV-1. PGDM1400 is one of three V2-targeting antibodies that are being developed, along with CAP256-VRC26.25  that combines the CD4bs specificity of VRC01-LS, PGDM1400 and the gp41 MPER binding of 10E8v4-variant, which is currently under clinical evaluation (NCT03705169). Viral replication and breakthrough viremia during treatment with VRC07-523LS, and CD4bs bNAbs in general, can occur despite substantial bNAb concentrations in serum and/or relatively low-level increase in neutralization resistance. Although the apparent lack of complete escape from VRC07-523LS neutralization in the presence of serum bNAb levels might suggest that such escape could be difficult for viruses to achieve, it also does not seem to be required to sustain robust plasma viremia. By maintaining higher levels of the bNAb, such as the suggested ID80 titers above the threshold of ~1,000, enhanced and perhaps longer-term control of viremia might be achieved; however, this would need to be confirmed in future clinical studies. Furthermore, another defining factor for the efficacy of bNAb combinations might be the pre-treatment level of viral replication. In our study, bNAbs were administered to viremic participants with up to ~160,000 HIV RNA copies per ml at the time of infusion. The antiviral activity and the ability to control virus more robustly might be enhanced in individuals with low-level viremia and/or low-level viral replication. Indeed, this was reported for macaques where PGT121 administration led to long-term SHIV control in animals with low baseline viral loads26, and we also previously observed that PGT121 suppressed viral replication for extended periods in a subset of participants with low baseline HIV RNA levels18. This, therefore, suggests, that although daily ART, and likely future long-acting antiretrovirals (ARVs), reliably suppress HIV in viremic individuals, the triple bNAb combination reported here might still perform well in individuals with low levels of replicating/reactivating virus—that is, what is seen during ATI, as reported by Mendoza et al.8 with a double bNAb combination. Indeed, such a strategy, testing a triple bNAb combination during ATI, is currently underway in an ongoing study (NCT03721510). Furthermore, in contrast to ARV medicines that must be continued indefinitely, bNAbs might have a significant advantage. Given their ability to target and mediate elimination of infected cells27 and to harness host immune responses29, a potential effect on reducing the size of the latent reservoir during bNAb therapy has been suggested30.Unfortunately, despite the extensive breadth of this bNAb combination, viral rebound in the presence of selected resistance mutations against PGDM1400 and PGT121 occurred rapidly in the studied participants. This might be explained, in part, by the potentially increased prevalence of PGDM1400 and PGT121 escape mutations in circulating viruses, as we previously reported31. Here as well, further optimization of bNAbs and bNAb regimens is necessary, and their role for prevention strategies, specifically with long-acting ARVs as a promising alternative, will need to be determined. Thus, combinations of ARVs and bNAbs, taking advantage of the synergistic effects of the single agents, are under active exploration (NCT03739996).Furthermore, combination bNAb therapy will likely be necessary for HIV prevention strategies, to provide broad coverage against the diversity of globally circulating viral strains. As recently reported in the Antibody-Mediated Prevention (AMP) study, the single bNAb VRC01 did not prevent overall HIV acquisition more effectively than placebo but was effective in preventing infection against VRC01-sensitive HIV isolates, suggesting that bNAb prophylaxis can be effective if the viral coverage is broad enoughThe present study has several limitations. The sample size for part 2 is small, with only three individuals available for complete virological analysis after triple bNAb therapy. The participants were not pre-screened for pre-existing bNAb resistance and/or selected based on viral susceptibility. Furthermore, participants, including participant 693–1969 who demonstrated the strongest virological response, were not tested for plasma ARV drug levels to confirm ART naivety.In summary, the results presented here will help to advance understanding of the capabilities, the limitations and the future role of bNAb combinations in HIV prevention and care. PGDM1400 could be included in future bNAb cocktails for passive immunization strategies, either for prevention or for therapeutic approaches given its complementary epitope specificity. Our data contribute to efforts to define a threshold bNAb(s) titer(s) that is required for viral control and that will inform dosing requirements and/or bNAb half-life engineering strategies for future monoclonal therapeutics.NCT03205917) In part 1, we evaluated three intravenous doses  of PGDM1400 given once or three intravenous doses  of PGDM1400 and three doses  of PGT121 given once  or in combination with PGT121  in HIV-uninfected adults at the Beth Israel Deaconess Medical Center (BIDMC) in Boston, Massachusetts  administration and could not be pregnant or breastfeeding. Participants were eligible for group 1 and group 2 if they were also 18–50 years of age and at low risk for HIV infection and willing to maintain low-risk behavior.Participants were eligible for the study across groups if they did not have any clinically significant acute or chronic medical condition (besides HIV), such as chronic hepatitis B, active hepatitis C, significant psychiatric disorder, alcohol or substance use disorder or chronic kidney or liver disease and if they had a body mass index >18 kg mParticipants with HIV (group 3) were eligible if they were 18–65 years of age, had CD4 ≥ 300 cells per μl, had no history of AIDS-defining illness within the previous 5 years and were not on ART for >6 months with detectable HIV-1 RNA levels between 1,000 and 100,000 copies per ml and (after appropriate counseling) were willing to defer ART treatment for at least 56 days after administration of the IP. All participants gave written informed consent and successfully completed an assessment of understanding before the initiation of study procedures.−1, 10 mg kg−1 or 30 mg kg−1) in group 1 and  in group 2 were randomized at a ratio of three antibody recipients to one placebo recipient, respectively . At each dose level in part 1, IP administration was separated by at least 24 hours for each of the first three participants. Randomization in part 1 ensured that at least two participants received active product and were observed for at least 24 hours before administration to additional participants. IP administration was also separated by at least 24 hours for each of the first three participants in part 2, group 3A, who received the triple bNAb combination.In part 1, eligible participants were enrolled first into the lowest dose subgroup of PGDM1400 alone (group 1A), and enrollment into the lowest PGDM1400 and PGT121 combination dose subgroup (group 2A) occurred only after the Protocol Safety Review Team (PSRT) reviewed the safety data through day 14 after administration of PGDM1400 alone and approved dose escalation. This staggered dose escalation was continued for each dose group. Participants in each subgroup were identified by a unique study identification number. Participants were randomized according to the randomization schedule prepared by the statisticians at the Data Coordinating Center  before the start of the study. Participants were automatically assigned a specific allocation number as they were enrolled into the data entry system. An unblinding list (Pharmacy List) was provided to the unblinded site pharmacist by the DCC. Study staff  and participants were blinded with respect to the allocation of the IP. A site pharmacist was unblinded for the purposes of preparing the IP. Blinded participants were informed about their assignment (product or placebo) at study completion, once the data were locked. As the bNAbs and placebo  looked identical in the infusion bag, no masking was required. In part 1, the four participants in each dose level subgroup ), in partial addition or flexible bags.Participants received the IP via intravenous infusion over approximately 60 minutes per antibody/placebo.+ T cell count were collected throughout the study for participants with HIV. Medical monitoring was provided by a PSRT and an independent Safety Monitoring Committee (SMC). Local and systemic AEs were graded by the NIAID Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events, version 2.1, July 2017. For the first 24 hours after IP infusion or injection, any infusion-related reactions, including cytokine release syndrome, were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0 (27 November 2017). Peripheral blood was collected to determine PGDM1400, PGT121 and VRC07-523LS serum levels, HIV sequencing and immunogenicity, among other research assessments outlined in the Study Protocol  = 23 RNA copies per ml), until it was replaced with the Hologic Aptima HIV-1 Quant Assay (LLoQ = 32 RNA copies per ml). HIV-1 VL was measured at OIC and HART using the Abbott Real-Time HIV-1 assay (LLoQ = 40 RNA copies per ml). VL assays were performed at LabCorp or at BIDMC. CD435. PGT121, PGDM1400 and VRC07-523LS concentrations were representative of three separate assays where each sample was run in duplicate. The standard curve consisted of PGT121 IgG monoclonal antibody, PGT121+PGDM1400 IgG monoclonal antibodies or PGT121+PGDM1400+VRC07-523LS IgG monoclonal antibodies, titrated in assay diluent, depending on subject group. The negative controls included an IgG1 monoclonal antibody (with specificity for an irrelevant antigen) and blank microspheres (uncoupled). Samples with bNAb concentrations below the LLoQ at a dilution of 1:50 were designated as the LLoQ value for plotting purposes. Samples with bNAb concentrations above the LLoQ at a 1:50 dilution were further tested at various dilution factors to obtain median fluorescent intensities (MFIs) in the linear range of the standard curve, and the resulting concentrations from the standard curve were averaged. Samples that were positive at or greater than the limit of detection (LOD) with an MFI that was three-fold over the pre-infusion MFI with a bNAb concentration less than the LLoQ were called positive. Samples with observed concentrations less than the LLoQ were called negative.(1) BAMA: PGT121, PGDM1400 and VRC07-523LS levels were determined on a Bio-Plex 200 system (Bio-Rad) that measures the ability of each monoclonal antibody to bind to their specific anti-idiotype antibody captured on fluorescent magnetic microspheres , using a customized and standardized HIV-1 assayLimits of quantification for analytes by HIV serostatus39. For participants in groups 1, 2 and 3B, neutralization titers were measured in pre-infusion and post-infusion immune sera against both viruses 6545.v4.c1 and THRO4156.18 (PGDM1400 sensitive and PGT121 resistant) and viruses X2088_c9 and CNE30 (PGDM1400 resistant and PGT121 sensitive). For participants in group 3A (triple bnAb combination), serum samples were tested using viruses 6540.v4.c1 , CH505TF.N334S.N160A.N280D.1 (PGT121 sensitive and VRC07-523LS and PGDM1400 resistant) and CAP220.2.00_A8_5B (VRC07-523LS sensitive and PGDM1400 and PGT121 resistant). MuLV was used as a negative control virus for all participant samples. Titers ranged from a minimum of 1:20 to a maximum of 1:1,562,500, with values outside of this range considered censored. The median IC50 titer of bNAbs against the specific indicator viruses used the clinical drug products tested at a primary concentration of 10 μg ml−1 with five-fold dilution series and were included in each individual assay setup. The estimated serum concentration for each individual bNAb was calculated as the serum ID50 titer × bNAb IC50 titer (μg ml−1).(2) TZM-bl neutralization assay: Neutralizing antibodies against HIV-1 were measured as a function of reduction in Tat-regulated luciferase (Luc) reporter gene expression in TZM-bl cells40. First-round polymerse chain reaction (PCR) was carried out with Platinum PCR Mix High-Fidelity (Invitrogen) together with HIV B primers listed in Supplementary Table 37.SGA assays were performed by isolating HIV-1 RNA and reverse transcribing to viral cDNAt1/2, clearance (CL/F), volume of distribution (Vz/F), area under the concentration decay curve (AUC) and effect of HIV RNA levels on PGDM1400 and/or PGT121 and/or VRC07-523LS disposition (elimination t1/2), CL/F, Vz/F and total exposure. The primary endpoint for antiviral activity among viremic participants with HIV was the change in plasma HIV-1 RNA levels from baseline . The secondary endpoints were change in CD4+ T cell count and frequency compared to baseline as measured by single-platform flow cytometry and development of HIV-1 sequence variations in epitopes known to result in reduced PGDM1400 and/or PGT121 and/or VRC07-523LS neutralization susceptibility. The primary endpoints for safety, tolerability and pharmacokinetics were changed in Protocol Version 4.0 to include the VRC07-523LS monoclonal antibody for the subgroup 3A (see The primary endpoints for safety and tolerability were as follows: (1) proportion of participants with moderate or greater reactogenicity  for 3 days after intravenous infusion of PGDM1400 alone, a combination of PGDM1400 and PGT121 bNAbs and a combination of PGDM1400 and PGT121 and VRC07-523LS; (2) proportion of participants with moderate or greater and/or PGDM1400- and PGT121- and VRC07-523LS bNAb-related unsolicited AEs, including safety laboratory  parameters after intravenous infusion of PGDM1400 and/or PGT121 and/or VRC07-523LS for the first 56 days after administration of the IP; and (3) proportion of participants with PGDM1400- and/or PGT121- and/or VRC07-523LS-related SAEs throughout the study period. The primary endpoints, for pharmacokinetics, were elimination n = 18). This was an exploratory proof-of-concept trial; the analysis was descriptive; and no formal null hypothesis was tested. The frequency of moderate or greater reactogenicity events was determined and compared among groups. The frequency of SAEs judged possibly, probably or related to the IP was determined. All AEs were analyzed and grouped by seriousness, severity and relationship to the IP (as judged by the investigators). An interim safety analysis of group data was carried out after each dose escalation according to the study schema without unblinding the study to investigators or participants. At the end of the study, a full analysis was prepared. Missing data were excluded from the statistical analysis. (2) The sample size for pharmacokinetic analysis was three per dose subgroup, sufficient for the planned analyses based on previous experience with PGT121 pharmacokinetics18. The data were fit to standard two-compartment population models using the Stochastic Approximation Expectation–Maximization (SAEM) estimation method in Monolix . Population (non-linear mixed effects) pharmacokinetic (popPK) models were fit separately by analyte and HIV infection status. Fixed effects were used to model the population-level pharmacokinetic parameters, and random effects were used to model the individual-level variability. The AUC was estimated by calculating the integral of the predicted concentration–time curve from the first infusion time to infinity. Additionally, peak concentration (Cmax) was computed as the maximum observed concentration. Summary descriptive results of pharmacokinetic parameters, including AUC, Cmax, t1/2 and CL/F results, were reported by bNAb and dose cohort. For each analyte, a Spearman correlation test was conducted to test for correlation among elimination t1/2, CL/F, Vz/F and dose- and weight-adjusted AUC with log10 VL at baseline  using mid-ranks for tied scores and the approximate distribution41. Correlation between pharmacokinetic and reported safety and pharmacodynamic outcomes were also explored parameters to examine exposure–effect relationships. The concordance correlation coefficient (CCC)42 was used to assess the concordance between the log10 concentrations from the binding and neutralizing antibody assays. (3) The sample size for virologic analysis was 6–18 participants across groups 3A and 3B. No placebo participants were enrolled in part 2 as per study design. For each participant, VL difference-from-baseline was defined as the difference in day 7 log10 plasma HIV-1 RNA levels from baseline . Based on a simulation study, power to reject the null hypothesis was 80% when the responder group has a difference-from-baseline VL drop of approximately 1.8 logs for a nominal α level of 0.05. As the study under-enrolled for group 3, the virologic outcome was not formally analyzed.(1) The sample size for safety and tolerability analysis was 30–66 participants according to the dose-escalation design used to characterize the safety profile of one intravenous infusion of PGDM1400 monoclonal antibody ± PGT121 monoclonal antibody, at one of three dose levels. For life-threatening AEs related to active product: if none of the nine (maximum 18) participants in either group 1 or group 2 who receive the active product experience such reactions, then the exact 95% upper confidence bound for the rate of these AEs in the population is 33.6% . Alignments were further refined manually. The bNAb resistance mutations . The phylogenetic tree for all participant viruses combined  using the default GTR model with site-wise rates and maximum likelihood optimization. Recombination analyses were performed using RAPR on the Los Alamos HIV database (https://www.hiv.lanl.gov/content/sequence/RAP2017/rap.html); additional details are in the legend for Extended Data Fig. https://www.hiv.lanl.gov/content/sequence/ANALYZEALIGN/analyze_align.html). Recombination analyses 43. Sequences are available at GenBank  / IC80. For baseline ID80 titers, day 7 bNAb concentrations were used. This was done to avoid the initially high bNAb concentrations in the serum immediately after infusion that rapidly decay as serum bNAbs seed tissues; this phase typically lasts 7 days. For rebound ID80 titers, the bNAb concentrations at the last HIV RNA level nadir time point were used, because this time point is the likely to be close to the time when rebound viruses start increasing in frequency. For bNAb combinations, ID80 titers were estimated as the factor by which the serum at a given time point with its composition of bNAbs will need to be diluted to give a predicted 80% neutralization in the pseudovirus neutralization assay. The fraction neutralization afforded by a given concentration profile for bNAbs in the serum was calculated using the Bliss–Hill model44 using serum concentration of bNAbs and individual bNAb IC50 and IC80 titers for each pseudovirus. Neutralizing activity  of bNAb combinations against global heterologous viruses  using data on individual bNAbs as available in CATNAP9.For individual bNAbs, ID50 and IC80 titers for each baseline and rebound viruses were obtained from previous studies for 3BNC117+10-1074 (ref. 4), 3BNC117 (ref. 3) and VRC01 (ref. 1) therapy in viremic patients. For each study, only those participants who showed a clear decline in the HIV RNA levels upon bNAb infusion were used; these are shown in Extended Data Fig. 80 titers were calculated as mentioned above.Individual bNAb concentrations and ICP values were obtained and are mentioned in the figure legends. RAPR, the web tool that was used for recombination analysis, employs the Wald–Wolfowitz Runs Test statistic, as described in Song et al.43.All group-based comparisons Fig.  were ana−1 each in viremic individuals with HIV not on ART (group 3A). These changes were made to allow safety, pharmacokinetic and antiviral activity evaluation of a triple bNAb combination, and primary, secondary and exploratory endpoints were updated accordingly. During this change, the number of participants in groups 3A and 3B was decreased from six (maximum 18) to three (maximum 9), thus changing the group 3 total from 12 (maximum 36) to six (maximum 18) and overall study total from 36 (maximum 84) to 30 (maximum 66). These changes were made to facilitate recruitment and with the approval of the SMC, the PSRT and the IRB.Protocol Version 4.0 added the VRC07-523LS monoclonal antibody to the study design in combination with PGDM1400 monoclonal antibody and PGT121 monoclonal antibody at 20 mg kgFurther information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-022-01815-1.Supplementary InformationSupplementary Tables 1–11, CONSORT 2010 checklist and protocol versionsReporting Summary


The proportion of HIV patients on second-line antiretroviral therapy is becoming a growing public health concern, especially in a low-income country setting. However, unlike first-line therapy, to date, very little is known about the outcomes of second-line therapy in the Ethiopia context. Thus, this study was conducted to determine the rate of treatment failure, death, and their predictors among HIV patients receiving second-line therapy.A retrospective cohort study was conducted on 642 people living with HIV in Dessie Comprehensive Specialized Hospital from October 2016 to November 2019. Poisson and competitive risk survival models were computed to explore predictors of treatment failure and death, respectively.During follow-up period, 39  of 568 patients had second-line treatment failure with 4.07 per 100 person-year rate of failure. Being on anti-TB treatment , not having optimal medication adherence [RR = 2.29 (95% CI: 1.09–4.78)], and not timely switched [RR = 5.89 (95% CI: 1.36–25.54)] were positively associated with treatment failure. Similarly, 44  of 642 patients died with 4.5 per 100 person-year rate of death. Being on advanced clinical condition , not having optimal medication adherence [SHR = 2.65 (95% CI: 1.31–4.74)], lower CD4 cell counts, and high viral load measurement were positively associated with death.A significant number of patients had failed to respond to second-line therapy. A large number of patients had also died. Patient medical profile and monitoring practice were associated with treatment failure and death. Hence, patient-centered monitoring and interventions should be strengthened, besides treatment switch. Globally, more than 26 million people living with HIV (PLWHIV) have accessed antiretroviral therapy (ART) by the end of 2020 , resultiHowever, unlike first-line therapy, to date, very little is known about the outcomes of second-line antiretroviral therapy; rate of treatment failure, and death in Ethiopia context. Previously, few studies have been conducted in Ethiopia on treatment failure , 9. HoweHIV patients receiving second-line therapy are expected to achieve viral re-suppression as well as immunological and clinical improvement . HoweverA retrospective cohort study was conducted at Dessie Comprehensive Specialized Hospital (DCSH) from October 2016 to November 2019. Dessie Comprehensive Specialized Hospital is located in the Amhara region, northeast Ethiopia, which is one of the top HIV burden areas in the nation . CurrentIn Ethiopia, the current standard second-line regimen consists of a combination of three ARV drugs (at least two of which are new to the patient); two Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and one Protease Inhibitor (PIs); Lopinavir/ritonavir (LPV/r) or Atazanavir /ritonavir (ATV/r) , 13. ThoSimilarly, in Ethiopia, PLWHIV data are handled by SMART care, ART registration (logbooks) and chronic ART follow up form (patient chart). The patient chart is the main source of data, which is filled by a trained health professional and includes detailed data elements. The SMART care electronic database is another source of data for patients on ART. It is filled by trained non-health professionals/data clerks/ by reviewing patient charts. SMART care has only a few key data elements like CD4, TB screen, and viral load. ART registration (logbook) is similar to SMART care, except it is filled manually.Antiretroviral treatment success is monitored using viral load (VL) measurement in which viral load measurement is done after initiation of ART at 6 months, 12 months, and every 12 months. In Ethiopia, viral load measurement was launched in 2016 on the top 20 burden areas including Dessie Comprehensive Specialized hospital , 11.Adult PLWHIV who have received second-line antiretroviral therapy at Dessie comprehensive specialized hospital is considered as the target population. Patients who were not taken second-line antiretroviral therapy for more than six months or those who had no viral load measurement after the commencement of second-line therapy were excluded from the analysis of treatment failure.The minimum representative sample size was calculated using EPI-Info StatCalc by taking AHR: 1.68 for a factor of TB-preventive (INH) had not taken, 18.41% of outcome in the unexposed group in the study done in Ethiopia . As wellThe primary outcome of this study was second-line treatment failure. A patient having two consecutive viral load measurements greater than or equal to 1000 copies/ mL with three months of Enhanced Adherence Support (EAS) confirms the failure of the current treatment regimen , 13. PatDeath was considered as a secondary outcome, which was confirmed by observing patient char and categorized as an event. Lost to follow-up is also defined as missing contact with the health care facility for more than three consecutive months. Patients who are alive on care or who have been transferred out, on the other hand, were considered censored.Age, sex, marital status, educational status, and disclosure status.3, viral load, first-line ART regimen before the switch, second-line ART regimen, medication adherence, and time between first enrolled to EAS and initiation of second-line therapy. Patients were considered timely switched if they shifted to second-line therapy within 3–6 months of enrollment in EAS . Patients who were on anti-TB treatment were 2.57 times more prone to second-line treatment failure as compared with counterparts [ARR = 2.57 (95% CI: 1.25–5.25)]. Similarly, patients who had not optimal medication adherence were 2.29 times more at risk of second-line treatment failure as compared with counterparts [ARR = 2.29 (95% CI: 1.09–4.78)] .In this study, 44 of 642 patients died with 4.5 per 100 person-year rates of death in 968.3 year observation period. The death rate was found by dividing the total number of deaths by the total follow-up period (i.e. 44/968.3 = 4.5 per 100 person). Out of total deaths, 23(52.2%) and 15(34%) deaths were reported at years one and two, respectively . During A competitive risk survival model showed that at therapy switch, patients who had an advanced clinical condition and lower CD4 cell counts were more at risk of death. After initiation of second-line therapy, patients who had not optimal medication adherence and viral load greater than or equal to 1000 copies/mL were more at risk of death.Patients who had advanced clinical conditions were 2.49 times more at risk of death as compared with the counterpart . Similarly, for every CD4 cell count increment, on average death rate decreased by 0.4%, while holding all other variables in the model remained constant [SHR = 0.996(95% CI: (0.994–0.999)].Patients who had not optimal medication adherence were 2.65 times more at risk of death as compared with counterparts . Similarly, PLWHIV who had a viral load greater than or equal to 1000 copies/mL were seven times more at risk of death as compared with counterparts, while holding all other variables in the model constant [SHR = 6.97 (95% CI: 3.3–14.74)] .Seven percent of PLWHIV had experienced second-line treatment failure during three follow-up periods. This finding is lower than studies conducted in the northwest and northern Ethiopia; 18.8% and 25.11%, respectively , 17. SimGenerally, this finding showed that a significant number of patients had experienced second-line treatment failure. This has both clinical and public health implications. Clinically, it increases the risk of drug resistance and the demand for high-cost third-line antiretroviral therapy. At the public level, it also increases the chance of HIV transmission, even resistant strain. Hence, due emphasis needs for HIV patients receiving second-line therapy, besides treatment switch.Being on anti-TB treatment and second-line treatment failure was positively associated, which is supported by studies conducted in Ethiopia and Uganda , 24. In A delay in switching after treatment failure was associated with second-line treatment failure. In line with this, studies done in Africa showed that delay to switch after first-line treatment failure leads to poor treatment outcomes –28. ThisRegarding the death of PLWHIV, seven percent of participants were died, of which more than fifty percent were reported with in the first year after the starting second-line therapy. This finding is lower than other studies, including a study done in Ethiopia –33. ThisHaving an advanced clinical condition and lower CD4 cell count at therapy switch was significantly associated with death, which is consistent with studies done in other settings , 34–37. Further, patients having poor medication adherence and high viral load  were also positively associated with the risk of death. This finding is in agreement with other studies –35. HencFirst, the study is the first of its kind in Ethiopia to provide first-hand information to improve the outcomes of patients on second-line therapy after the implementation of the viral load test. Second, it was conducted on a large sample size as well as used different data sources  that increases consistency as well as minimize incompleteness.The finding of this research can be also extrapolated in other low-income countries’ settings where they implement similar WHO treatment modalities, outcome ascertainment, and have a similar socio-demographic contexts .This study failed to collect and analyze data on behavioral, social, and psychological factors as well as facility-level determinants of treatment failure and death. Those limitations were related to the retrospective cohort design, in which data were collected by reviewing medical charts Furthermore, this study was unable to get and consider other clinical profiles such as organ function results, drug resistance tests. Therefore, confounding through the unmeasured covariates need to be considered while interpreting the reported associations. Another limitation of this study was fail to report the specific cause of death.A large percentage of patients had not responded to second-line treatment. A substantial proportion of the patients had also died. Treatment failure and death were linked to the patient’s medical profile and monitoring practices. As a result, in addition to treatment switching, patient-centered monitoring and treatments should be reinforced.S1 File(DOCX)Click here for additional data file.


To date, therapeutic switches are performed to reduce and prevent toxicity, improve adherence, promote virological control, and save costs. Drug switches are a daily challenge in the management of people living with HIV (PLWH), especially in those with multiple comorbidities and on polypharmacy. The objectives of this prospective analysis were: (I) to evaluate the viro-immunological efficacy of BIC/FTC/TAF in a cohort of PLWH who switched to this regimen from any other previous, at the Infectious and Tropical Diseases Unit of the Padua University Hospital; (II) to assess the impact on body weight, lipids, and renal function parameters at week 48; and (III) to evaluate daily costs changes, adherence, and the rate and causes of discontinuation of the regimen. We included all adult PLWH who switched to BIC/FTC/TAF from 1 February 2020 to 31 October 2021. We collected demographic, clinical, and laboratory data at baseline and week 48 after the switch. In addition, the estimated cART-related cost changes over the follow-up period were calculated. Over the study period, 290 individuals who switched to BIC/FTC/TAF, 76.9% were males, with a median age of 52 years, and 94.8% had an undetectable baseline HIV viremia. After a median time of 35 days (IQR: 1–55), 41 (14.1%) individuals discontinued the regimen. Factors significantly associated with discontinuation were switching from dual regimens, and neurological disorders. At week 48, we detected a significant increase in body weight, BMI, CD4 T-cell count, and CD4/CD8 ratio, and a significant reduction in triglycerides and costs; all patients had undetectable HIV RNA. Our results showed that switching to BIC/FTC/TAF may favor slightly immunological recovery and cost saving . The reduction in triglycerides does not appear to be clinically relevant, even if statistically significant, nor do both the increase in body weight and BMI  and the increase in CD4 T-cell count (+45 cells/mmc). Further studies are needed to confirm our results. The introduction of combination antiretroviral therapy (cART) revolutionized the natural history of HIV disease, allowing people living with HIV (PLWH) to reach a life expectancy overlapping that of the general population ,2. Over On the other hand, the use of this class remains preferable in subjects with multiple resistance mutations to classes of nucleoside inhibitors (NRTIs) and non-nucleoside (NNRTI) of reverse transcriptase . INSTIs,The main objective of this prospective study was to evaluate the viro-immunological and clinical efficacy of the BIC/FTC/TAF switching regimen in a cohort of individuals with HIV-1 infection attending the Infectious and Tropical Diseases Unit of the Padua University Hospital (from baseline to week 48). Secondary objectives were: (i) to assess the impact on body weight, lipids , and renal function parameters (from baseline to week 48); and (ii) to evaluate the level of adherence, the rate and causes of the discontinuation of this regimen, and the costs.In this mono-centric prospective observational cohort study, we included all consecutive PLWH older than 18 years of age who switched to BIC/FTC/TAF combination from any previous dual or 2NRTIs-backbone-based antiretroviral regimen already ongoing for at least 6 months and regardless of plasma viremia. Individuals’ recruitment started on 1 February 2020, and ended on 31 October 2021, at the Infectious and Tropical Diseases Unit of Padua University Hospital .We collected information on demographic , and clinical and HIV-related parameters . As per the study objective, the following information was collected at baseline (before BIC/FTC/TAF initiation) and 48 weeks after the switch: plasma HIV-RNA (virologic suppression or undetectability was defined as a limit of HIV RNA detection: <20 copies/mL), CD4+ T-cell count, CD4/CD8 ratio, creatinine, estimated glomerular filtration rate (eGFR), glucose, total cholesterol, triglycerides, HDL, LDL, body weight (kg), height (cm), and body mass index (BMI). Individuals were categorized by BMI class according to WHO definitions . Undetecp-values < 0.1. The analyses were run through Stata SE 16 .Continuous and categorical variables were reported as median value (interquartile range) and absolute number (proportion), respectively. The intra-subject median changes from baseline to week 48 were analyzed by paired Wilcoxon test after excluding subjects classified as discontinuations; significance for changes in binary and multinomial variables was analyzed by the symmetry McNemar chi-square test and by the marginal homogeneity test, respectively. Inter-group comparisons were performed by Kruskal–Wallis test and chi-square test for trend. Binary and multinomial logistic regression was considered for univariate The study protocol received approval from the local Ethics Committee (n. 4052AO17) and written informed consent was obtained from each participant prior to enrollment in the study. In addition, the study was conducted accordingly to principles of good clinical practice and to the Helsinki declaration.Over the study period, 290 individuals started the BIC/FTC/TAF. As reported in At week 48, the intention-to-treat (ITT) analysis showed that the BIC/FTC/TAF regimen warranted virologic suppression in 95% of individuals; while in the per-protocol analysis population, no patient had HIV-1 RNA > 20 copies/mL at the end of follow-up, including those subjects 14 (5.6%) who were on virological failure before the switch. No discontinuation of the regimen due to virological failure was observed during the study period.p < 0.001). A questionnaire on self-reported antiretroviral adherence did not show any differences from baseline to the follow-up time point (The median CD4+ T cell count significantly increased from 568 (IQR: 400–756) cells/L at baseline to 610 (IQR: 450–780) cells/L after 48 weeks of follow-up. A statistically significant increase in CD4+/CD8+ ratio was also detected, from 0.72 (IQR: 0.48–1.16) to 0.81 (IQR: 0.56–1.19) (me point .p = 0.057). Univariate and multivariate results are shown in p = 0.015 compared to switching from an INI-based regimen) and with the presence of neurological disorders, independently from age, sex, ethnicity, HCV serostatus, polypharmacy, and psychiatric disorders.Overall, 41 individuals (14.1%) discontinued the study regimen: 15 (5.2%) individuals for toxicity/intolerance, 6 (2.1%) subjects who were previously pro-actively simplified to a dual regimen containing dolutegravir and lamivudine, 1 (0.3%) subject for weight gain greater than 5% from the baseline, 2 (0.7%) subjects for both weight gain and intolerance, 2 (0.7%) women because of pregnancy, and 15 (5.2%) individuals who were lost to follow-up. In subjects who discontinued the regimens for toxicity, the median time from switch to discontinuation was 35 days (IQR:1–55). Adverse events causing discontinuation  were gastrointestinal disorders , sleep disturbances , neuropsychiatric disorders , elevation of liver enzymes , difficulty to swallow the tablet , itching , myalgia , and erectile disfunction , with four subjects experiencing more than one adverse event. When we compared individuals who maintained the regimen with those who continued it , we deteBIC/FTC/TAF is one of the latest STRs introduced in clinical practice including a high genetic barrier integrase strand transfer inhibitor with TAF/FTC. It is recommended by most antiretroviral therapy guidelines both as an initiation and switch regimen in PLWH. Even though its virological efficacy was demonstrated in large double-blind randomized clinical trials, prospective real-life data assessing durability, impact on viro-immunological, clinical, metabolic parameters, and costs of BIC/FTC/TAF prescribed as switch regimen are still scarce. In our cohort in an analysis, the switch to BIC/FTC/TAF was associated with an overall maintenance of virologic suppression in 95% of individuals, and no virological failure in the 249 subjects who completed the 48 weeks of follow-up was observed. No patients discontinued the regimen due to VF during the time of observation, and viral suppression was maintained also in individuals with virological failure at baseline regardless of the pre-existing NRTI resistance such as M184V and K65R. These results further support the high resistance barrier of bictegravir as previously demonstrated in both in vitro and clinical studies ,24,25. IIn clinical trials performed on naïve individuals, which reached 5 years of follow-up, the rate of discontinuation was around 11–13% , occurriIn the multivariate analysis, neurological disorders were significantly associated with drug discontinuation. Indeed, it is well known that central nervous system (CNS)-related adverse events have been associated with the use of INSTI-based treatment, including DTG, for which the incidence of insomnia and headache ranged from 3% to 15 ,29,30. MOf note, the long HIV history of our patients associated with the large variety of previous antiretroviral agents precluded a meaningful description of the role bictegravir may have had on CNS-related AEs. Interestingly, none of the patients who withdrew BIC/FTC/TAF for CNS-related AEs were older than 50 years old, confirming good tolerability of the regimen in aging people (data not shown).We believe that in our study the higher proportion of participants who withdrew because of adverse events may be somewhat related to the COVID-pandemic period during which the study was conducted. It is known how COVID-19 had an important impact on the “HIV world” , and it is still also debated whether people with HIV have a poorer prognosis than the general population ,33,34. TAmong individuals who were considered as discontinuation, the loss to follow-up rate  was also very similar to the one reported by clinical trials in naïve individuals. However, in our cohort, most lost to follow-up individuals were black-African people (migrant status) who represented about 20% of the study population. This finding is not surprising considering that in our region black people are often migrants, and a difficult-to-treat population, with low levels of adherence and high rates of loss to follow-up. Furthermore, we believe that these results draw us closer to the unmet needs of this subgroup of people to which we should likely guarantee a better linkage to care. In this specific population, the efficacy, tolerability, and lack of resistance to BIC/FTC/TAF may represent a good treatment option.Switching to BIC/FTC/TAF did not result in a significant reduction in total cholesterol, LDL cholesterol, and triglycerides in our cohort . In prevIn our study most individuals at baseline were treated with INSTI and TAF; therefore, it is reasonable that switching to BIC/FTC/TAF had a “neutral” lipidic effect, even if in the absence of the known lipid-lowering effects of TDF compared with TAF and in the absence of lifestyle modifications.In fact, in our study, we explored for the first time, the changes/lifestyle modifications  from baseline to follow-up.Indeed, the failure to change the lifestyle  may have a role in the significant increase in BMI in our cohort of individuals. At month 12 of follow-up, a mean increase of +1 kg was detected and 17% of individuals showed a significant increase in the BMI value. Even though the weight gain observed in our study overlaps that foreseen by the WHO in the general population , these rSecondly, it is likely that the study regimen does not imply a clinically relevant weight gain over 12 months. It is interesting to note that in our population about 38% of individuals were overweight, with 12% obese at study enrollment. These data are shared with other large HIV cohorts, such as the “Opera” cohort where among a total of 6908 virological suppressed PLWH, overweight (BMI ≥ 25 to <30) and obesity (BMI > 30) were reported in 37–39% and 26–29% of participants, respectively . It is wIt is difficult to speculate on a possible evolution of all the different parameters over time. However, it is likely that patients may put further weight on during a longer follow-up; from of a mean 1 kg/year as already shown in the general population, to 5–6 kg during 5 years of follow-up as already observed in the above-mentioned clinical trials on INSTIs combined with tenofovir alafenamide.However, further studies are needed to evaluate the mechanism of weight gain among individuals starting INSTI-based regimens. In the meantime, clinicians have to be aware that the development of overweight and obesity in the HIV population may significantly increase the risk of cardiovascular events and should be prevented. In this context, the robustness and the convenience of the BIC/TAF/FTC regimen may be integrated into care interventions and counseling oriented towards a healthy lifestyle.Finally, given the economic impact of costly brand antiretroviral treatments in high-income countries, the BIC/TAF/FTC regimen in our cohort of individuals allowed an overall amount of saving of 1533 EUR/patient/year; it was not a budget-impact analysis but only/simply a direct analysis of the daily cost of the regimen. However, we obtained the result of a successful regimen without de-simplifying a single tablet antiretroviral combination, often considered a cost-saving strategy, and without compromising the best quality of care. This aspect needs to be considered in terms of resource-saving, allowing clinicians to consider not only the medical concerns but also cost-saving strategies, warrantying safe switches at a lower cost.Unfortunately, our study is somewhat limited by the low numbers, and by the high rates of individuals who were lost to follow-up. Moreover, we assessed only the raw cost of switching, without performing a proper cost–benefit analysis. In conclusion, in our study, BIC/FTC/TAF has been demonstrated to be an excellent option for switching therapy in HIV people presenting in different clinical contexts and in individuals presenting with metabolic disorders.