Limited cross-variant immune response from SARS-CoV-2 Omicron BA.2 in naïve but not previously infected outpatients

Summary Omicron is currently the dominant SARS-CoV-2 variant and several sublineages have emerged. Questions remain about the impact of previous SARS-CoV-2 exposure on cross-variant immune responses elicited by the SARS-CoV-2 Omicron sublineage BA.2 compared to BA.1. Here we show that without previous history of COVID-19, BA.2 infection induces a reduced immune response against all variants of concern (VOC) compared to BA.1 infection. The absence of ACE2 binding in sera of previously naïve BA.1 and BA.2 patients indicates a lack of meaningful neutralization. In contrast, anti-spike antibody levels and neutralizing activity greatly increased in the BA.1 and BA.2 patients with a previous history of COVID-19. Transcriptome analyses of peripheral immune cells showed significant differences in immune response and specific antibody generation between BA.1 and BA.2 patients as well as significant differences in the expression of specific immune genes. In summary, prior infection status significantly impacts the innate and adaptive immune response against VOC following BA.2 infection.

The ability of BA.2 infection to induce neutralizing antibodies in unvaccinated individuals, either without or with previous SARS-CoV-2 infection, is pending definition. Here we conducted a comparative investigation of the innate and humoral immune response elicited in 74 Omicron outpatients infected with BA.1 (Lee et al., 2022b) or BA.2 that were either naive prior to Omicron infection or had prior exposure to other SARS-CoV-2 variants. We measured antibody titers and ACE2 binding inhibition as a proxy for neutralization. In addition, we investigated the immune transcriptome and the repertoire of antibody germline alleles in these cohorts.

Antibody response after omicron subvariant infection
We measured antibody titers and neutralizing antibody responses in 74 outpatients infected with either Omicron BA.1 or BA.2 sublineages. Demographic and clinical information are provided in Table 1. Although none of the patients had been vaccinated, 24 had been infected by an earlier variant. More than 20% of the BA.1 infected, but none of the BA.2 infected patients, had moderate or severe disease.
Humoral immune response was measured between 7 and 21 days (median: days 13-14) post-documented Omicron BA.1 or BA.2 infection. In previously uninfected and unvaccinated individuals, anti-Omicron BA.1 spike IgG titers were approximately 40-fold lower following BA.2 infection compared to BA.1 infection ( Figure 1A; Table S1). IgG titers against the ancestral spike were almost 70-fold lower following BA.2 as compared to BA.1 infection. Anti-spike IgG antibody titers in individuals previously infected with earlier SARS-CoV-2 variants were at least 5-fold higher with no difference in titers between those infected with the BA.1 versus BA.2 variants ( Figure 1A; Table S1). This highlights that previous infection with variants has a particularly strong impact on the humoral immune response in BA.2 infected patients.
For reference, titers from individuals infected with the Omicron Beta variant  are shown. These approximate those obtained in individuals previously infected with an earlier variant who then experienced BA.1 or BA.2 infection. The differential response in antibody generation following BA.1 and BA.2 infection was consistent across all SARS-CoV-2 variants tested ( Figure 1B). We next measured neutralization capacity using the angiotensin-converting enzyme 2 (ACE2) binding inhibition assay ( Figure 1C). Neutralizing activities against the ancestral strain and Omicron BA.1 were similarly low in antigen-naive individuals infected with either BA.1 or BA.2. Like the overall antibody titers, iScience Article neutralizing activity was higher in those previously infected. Activity from BA.1 and BA.2 infected individuals with previous SARS-CoV-2 infection approximated that found for individuals infected with the Omicron Beta variant and was similar across all SARS-CoV-2 variants tested against ( Figure 1D).

Transcriptome response
Next, we investigated the impact of BA.1 and BA.2 infection on the immune transcriptome in Omicron patients with or without a previous infection history ( Figure 2; Table S2: Significantly differential genes between BA.1 and BA.2 infected cohorts with no prior infection; Table S3: Significantly differential genes between no prior and prior infection in BA.2 infected cohorts). Buffy coats were isolated within the first three days after validated Omicron infection and bulk RNA-seq was conducted with an average sequencing depth of 200 million reads per sample. First, we directly compared the transcriptomes of the BA.1 and BA.2 infected Omicron cohorts without prior infection. In a direct comparison of the two cohorts, expression of 38 and 15 genes was induced significantly in patients infected with BA.1 and BA.2, respectively ( Figure 2A; Table S2: Significantly differential genes between BA.1 and BA.2 infected cohorts with no prior infection). Although genes activated upon BA.1 infection are enriched in neutrophil degranulation and innate immune system, the ones activated upon BA.2 infection are categorized in complement pathway ( Figure 2B; Table S2: Significantly differential genes between BA.1 and BA.2 infected cohorts with no prior infection; Figure S1). Genes induced at significantly higher levels in BA.1 patients, such as C4BPA, initially identified by RNA-seq analyses ( Figure 2C) and validated by qRT-PCR ( Figure S1A), have also been reported to be regulated in COVID-19 patients (Gutmann et al., 2021;Jabeen et al., 2022). Of note, the complement genes C1QA and C1QB that show elevated expression in nonclassical monocytes from COVID-19 patients (Stephenson et al., 2021) are preferentially expressed in BA.2 patients as initially identified by RNA-seq analyses ( Figure 2C) and validated by qRT-PCR ( Figure S1B). We then compared the immune transcriptomes from BA.2 patients with and without prior infection and identified 342 and 145 significantly induced genes, respectively ( Figure 2D; Table S3: Significantly differential (C and D) Neutralization response to virus spike protein of the ancestral strain and Omicron variants (C) as well as the SARS-CoV-2 RBD (spike) of former variants (D). p-value between the two groups is from the one-tailed Mann-Whitney t-test. Percentage of samples with zero inhibition activity for each group is shown above the X axis. Asterisks indicate significance between groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Line at median, dotted line at 90%.

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iScience 25, 105369, November 18, 2022 3 iScience Article genes between no prior and prior infection in BA.2 infected cohorts). GSEA analyses linked the induced genes in the ''no prior infection'' cohort to innate immune responses, including interferon and inflammatory pathways ( Figure 2E). Genes activated in the ''no prior infection'' cohort such as CCL2 and IL6, initially identified by RNAseq ( Figure 2F) and validated by qRT-PCR ( Figure S1C), are part of the interferon response in severe COVID-19 (Cao, 2021;Munnur et al., 2021). Similar to what has been previously reported for transcriptome analysis of peripheral blood mononuclear cells of SARS-CoV-2 infected patients, in general, there was good agreement between RNA-seq ( Figure 2) and qRT-PCR results for genes differentially expressed between different cohorts ( Figure S1) (Zhang et al., 2021b).

Antibody germline repertoire
The higher level of anti-spike antibodies and neutralization activity in BA.1 patients of the ''no prior infection'' group compared to BA.2 infection, led us to dig deeper and interrogate the germline antibody gene usage. Specifically, we determined the range of immunoglobulin heavy chain (IGHV) gene usage elicited by BA.1 and BA.2 sublineage infection in the ''no previous infection'' cohort ( Figure 3). RNA-seq was conducted 13-16 days after the positive PCR test. The number of total immunoglobulin genes (heavy chain variable (IGHV), light chain variable (IGKV and IGLV) and T cell receptor alpha and beta variable (TRAV and TRBV)) revealed the use of a broad range of germlines in all cohorts without statistically significance ( Figure 3A). However, we observed an increased frequency of transcription of eight VH genes, including IGHV3-11,

DISCUSSION
While the Omicron BA.1 and BA.2 sublineages share mutations they also carry unique mutations, 28 in BA.2, which can explain their differential response to vaccine-induced and therapeutic antibodies (Ai et al., 2022;Arora et al., 2022aArora et al., , 2022bArora et al., , 2022b. Previous studies have also demonstrated that BA.1 infection of naive persons yields a very limited antibody response to SARS-CoV-2 VOCs other than Omicron itself (Lee et al., 2022b;Rö ssler et al., 2022). However, prior vaccination or prior infection with older SARS-CoV-2 variants augment the immune response upon BA.1 breakthrough infection (Lee et al., 2022b;Rö ssler et al., 2022). Our study now reveals that in antigen-naive individuals the immunologic response following BA.2 infection is even lower than that elicited by BA.1 infection as indicated by significantly lower antibody titers against all VOCs.
While other studies have focused on the humoral response including neutralizing activity induced by Omicron infection (Suryawanshi et al., 2022), we have extended the investigations and explored the presence of germline immunoglobulin variants induced by BA.1 and BA.2 infection. RBD-targeting antibodies can block SARS-CoV-2 binding to ACE2 and neutralizing immunoglobulin G heavy-chain variable (IGHV) gene usage has been reported (Yuan et al., 2020). Our study identified several IGHV germline alleles in Omicron patients that have been identified in neutralizing antibodies in COVID-19 patients (Yan et al., 2021;Yuan et al., 2020).
In conclusion, our study demonstrates that Omicron BA.2 infection of unvaccinated individuals without prior COVID-19 does not elicit effective antibodies against BA.1 and non-Omicron variants. In previously infected individuals, however, BA.2 infection successfully induces immunity against other variants.

Limitations of the study
There are several limitations to the current study. First, the study was conducted on volunteers from a specific geographical area, Tyrol (Austria). Secondly, the limitation is the confinement of the study to a timeframe of three weeks following Omicron infection. Third, the number of BA.2 patients with prior infection was very limited. Additional individuals for this cohort could not be recruited due to the replacement of BA.2 infection by BA.4 and BA.5 in Tyrol. Similar pandemic-related cohort size limitations have been noted for other transcriptome-based COVID studies (Sureshchandra et al., 2021;Zhang et al., 2021b). Lastly, some of the data collected on breakthrough infections were reliant on retrospective chart review and not collected as part of a prospective study.   Our gratitude goes to the participants who contributed to this study to advance our understanding of SARS-CoV-2 Omicron infection. We thank Yuhai Dai from the NIDDK clinical core for helping antibody assay. This work was utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih. gov). RNA-sequencing and single-cell RNA-sequencing were conducted at the NIH Intramural Sequencing Center, NISC (https://www.nisc.nih.gov/contact.htm).

DECLARATION OF INTERESTS
The authors declare no competing interests.

INCLUSION AND DIVERSITY
We worked to ensure gender balance in the recruitment of human subjects. We worked to ensure ethnic or other types of diversity in the recruitment of human subjects. We worked to ensure that the study questionnaires were prepared in an inclusive way. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper self-identifies as a gender minority in their field of research. One or more of the authors of this paper self-identifies as living with a disability. While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list. We avoided ''helicopter science'' practices by including the participating local contributors from the region where we conducted the research as authors on the paper. iScience 25, 105369, November 18, 2022 iScience Article followed the 'Sex and Gender Equity in Research -SAGER -guidelines' and included sex and gender considerations where relevant.

Antibody assay
Whole blood was collected by medical personnel after subjects had tested positive for SARS-CoV-2. Antibody containing sera were obtained by centrifuging EDTA blood samples for 10 min at 4,000 g. End-point binding IgG antibody titers to various SARS-CoV-2-derived antigens were measured using the Meso Scale Discovery (MSD) platform. SARS-CoV-2 spike, nucleocapsid, Alpha, Beta, Gamma, Delta, and Omicron spike subdomains were assayed using the V-plex multispot COVID-19 serology kits (Panel 23 (IgG) Kit, K15567U). Plates were coated with the specific antigen on spots in the 96 well plate and the bound antibodies in the samples (1:50,000 dilution) were then detected by anti-human IgG antibodies conjugated with the MSD SULPHO-TAG which is then read on the MSD instrument which measures the light emitted from the tag.

ACE2 binding inhibition (neutralization) ELISA
The V-PLEX COVID-19 ACE2 Neutralization kit (Meso Scale Discovery, Panel 23 (ACE2) Kit, K15570U) was used to quantitatively measure antibodies that block the binding of ACE2 to its cognate ligands (SARS-CoV-2 and variant spike subdomains). Plates were coated with the specific antigen on spots in the 96 well plate and the bound antibodies in the samples (1:10 dilution) were then detected by Human ACE2 protein conjugated with the MSD SULPHO-TAG which is then read on the MSD instrument which measures the light emitted from the tag.

Extraction of the buffy coat and purification of RNA
Whole blood was collected, and total RNA was extracted from the buffy coat and purified using the Maxwell RSC simply RNA Blood Kit (Promega) according to the manufacturer's instructions. The concentration and quality of RNA were assessed by an Agilent Bioanalyzer 2100 (Agilent Technologies, CA). mRNA sequencing (mRNA-seq) and data analysis The Poly-A containing mRNA was purified by poly-T oligo hybridization from 1 mg of total RNA and cDNA was synthesized using SuperScript III (Invitrogen, MA). Libraries for sequencing were prepared according to the manufacturer's instructions with TruSeq Stranded mRNA Library Prep Kit (Illumina, CA, RS-20020595) and paired-end sequencing was done with a NovaSeq 6000 instrument (Illumina) yielding 200-350 million reads per sample.
For T-or B-cell receptor repertoire sequencing analysis, trimmed fastq files from bulk RNA-seq were aligned against human V, D and J gene sequences using the default settings with MiXCR (Bolotin et al., 2015(Bolotin et al., , 2017.

QUANTIFICATION AND STATISTICAL ANALYSIS
Differential expression gene (DEG) identification used Bioconductor package DESeq2 in R. P-values were calculated using a paired, two-side Wilcoxon test and adjusted p-value (pAdj) corrected using the Benjamini-Hochberg method. The cut-off value for the false discovery rate was pAdj >0.05. Genes with log 2 fold change >1 or < -1, pAdj <0.05 and without 0 value from all sample were considered significant. For significance of each GSEA category, significantly regulated gene sets were evaluated with the Kolmogorov-Smirnov statistic. p-values of antibody and qRT-PCR between two groups were calculated using one-tailed Mann-Whitney t-test on GraphPad Prism software (version 9.0.0). A value of *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 was considered statistically significant.