scrub typhus of tsutsugamushi disease is an acute , febrile disease caused by infection with orientia tsutsugamushi . o . tsutsugamushi is a gram - negative bacterium but has neither lipopolysaccharide nor a peptidoglycan layer . orientia isolates are highly variable in their antigenic properties . the major surface protein of o . tsutsugamushi is the variable 56 - kda protein . serotype - specific monoclonal antibodies to orientia react with the 56 - kda protein . although sera from most patients with scrub typhus recognize this protein , another 47 - kda protein is also recognized , suggesting that both 56 - kda and 47 - kda proteins are good candidates for use as diagnostic antigens and that the combination of these two proteins has potential use as an immunogenic composition . in accordance with the present invention , there is generally provided a scrub typhus immunogenic composition comprising an r47 truncated protein of seq id no . 1 , an r56 truncated protein ; and a pharmaceutically acceptable carrier . the 47 kda antigen is a membrane protein . in order to produce a properly folded r47 protein in aqueous solution , its n - terminus which contains the hydrophobic domain was truncated . forward primer seq id no . 5 and reverse primer , seq id no . 6 , were designed to amplify the gene segment of nucleotides 174 - 1481 , seq id no . 7 . the amplified gene segment was cloned into pet24a vector . the expressed protein sequence is in seq id no . 1 . in embodiments of the inventive subject matter , the scrub typhus immunogenic composition may comprise the r47 truncated protein in combination with one of the following r56 truncated proteins : seq id no . 2 , seq id no . 3 , or seq id no . 4 and a pharmaceutically acceptable carrier . the optimum dose of the immunogenic composition is contemplated to be 0 . 5 - 20 mg for humans and 0 . 5 - 2 . 5 mg for primates . administration of the immunogenic composition is done subcutanously . the inventive subject matter also provides an assay for detecting antibody to scrub typhus comprising obtaining a sample from a subject , exposing the sample to a truncated r47 protein as represented in seq id no . 1 in assay equipment . such assay equipment may be selected from the group consisting of elisa plates , dot - blot matrices , and hand held chromatographic and flow through assay devices . samples suitable for such assay methods include blood , oral fluids and urine from which antibodies could be detected and from which antigens could be detected by way of laboratory generated antibodies . the inventive subject matter further comprise a method of treating a patient suffering from scrub typhus , the method comprising administering a pharmaceutically effective dose of antibodies raised against the combination of truncated proteins of seq id no . 1 and the truncated r56 protein selected from the group consisting of seq id no . 2 , seq id no . 3 and seq id no . 4 and a pharmaceutically acceptable carrier . the inventive subject matter also contemplates a pharmaceutical composition for treatment of an hiv patient comprising antibodies raised against a truncated r56 protein and a pharmaceutically acceptable carrier wherein administration of the composition reduces the patients viral load . such truncated proteins may be selected from the group consisting of seq id nos . 2 - 4 or combinations thereof . the dosing and administration are the same as stated above for the immunogenic compositions . the present inventive subject matter further contemplates a treatment of an hiv patient which comprises antibodies raised against a truncated r47 protein selected from the group of seq id no . 1 and a pharmaceutically acceptable carrier which results in a reduction of the hiv viral load of an hiv patient . dosing and administration are also as stated above for this embodiment . the inventive subject matter also contemplates treatment of an hiv patient which comprises administering a pharmaceutical composition comprising antibodies raised against a combination of an r47 truncated protein and a truncated r56 protein and a pharmaceutically acceptable carrier to reduce the patients hiv viral load . dosing and administration are also as stated above for this embodiment . the inventive subject matter also contemplates an immunogenic composition against hiv infection comprising a pharmaceutical effective dose of antibodies raised against a truncated r47 protein and a truncated r56 protein of seq id nos . 2 - 4 and combinations thereof . dosing and administration are as stated above for this embodiment . the following examples are illustrative of preferred embodiments of the invention and are not to be construed as limiting the invention thereto . forward and reverse dna primers ( seq id no . 5 - 6 , respectively ) specific for 47 - kda ( accession no . l31934 . 1 ) were designed with bamh1 and ncoi ( both from neb ) restriction sites and used for pcr to amplify the gene coding for a truncated 47 - kda protein using dna purified from karp strain of o . tsutsugamushi . the pcr product ( insert ) was ligated to plasmid pet24d ( novagen ) digested with bamh1 and ncoi . the sequence of insert was confirmed and the ligated plasmid was transformed into bl21 ( de3 ) cells ( invitrogen ). colony containing ligated plasmid was grown 2yt medium and induced with 1 mm of iptg at 37 ° c . for 3 h for protein expression . to purify this expressed protein , 600 ml of b121 ( de3 ) cells were grown , induced , centrifuged and cell pellet was lysed with 20 mm tris - hcl ( ph 8 . 0 ) containing 5 mm edta and 1 mm pmsf according to the method developed by ching et al ., clin . diag . lab . immuno . 5 , 519 - 526 , 1998 . after ultrasonic disruption and centrifugation , the expressed protein was mainly associated with supernatant rather than inclusion body . this fraction was further purified with a 30 , 000 mw cutoff membrane follow by deae anion exchange using 20 mm tris ph 8 . 0 and 2m nacl salt gradient ( agilent 1100 hplc ). alternatively , the protein was separated on a deae anion exchange using 20 mm tris ph 7 . 0 and 2m nacl salt gradient . the protein eluted with salt gradient was collected , analyzed with sds - page , and stained with coomassie blue to identify the purity of each fraction . in some cases , different fractions were poured together for the sds - page analysis . the sequence homology of 47 - kda protein with other known proteins was searched with standard blast method and the conserved domain analysis of 47 - kda protein was analyzed using ncbi cdd algorithm . ( fig5 - 6 ) in addition , proteins with similar domains as 47 - kda protein were aligned together to analyze the conserved amino acid sequence within the family . the dna coding for 47 - kda protein was successfully pcr amplified using dna purified from o . tsutsugamushi karp strain as template and primers designed specifically for this genes ( fig1 ). the cloning of this pcr product into pet24d (+) plasmid was confirmed after digestion with bamh1 and ncoi ( fig1 ) and dna sequence was verified with a dna sequencer . expression of the 47 - kda protein was observed after 3 h of 1 mm iptg induction at 37 ° c . ( fig2 ). the sequence of this expressed protein was confirmed with protein sequencer . ( after sonication and centrifugation , the expressed protein was mainly associated with supernatant after the first centrifugation ( fig2 ) rather than associated with inclusion body as the r56 protein reported by ching et al . the expressed protein may be associated with the membrane fraction and the addition of detergent ( 1 % of np40 ) appears to increase the yield during purification procedure ( data not shown ). part of the supernatant containing r47 was first purified with deae anion exchange on a hplc using a tris buffer at ph 8 . 0 and protein was eluted with a nacl gradient ( up to 0 . 8 mm ) and the fractions were collected and analyzed with sds - page ( fig3 ). the other part of the supernatant was purified with hplc using a tris buffer at ph 7 . 0 and eluted with a nacl gradient ( up to 2 m ). the resulted fractions were poured and analyzed for r47 ( fig4 ). the search of similarity of r47 with standard blast resulted in homologues with various serine proteases ( fig5 ). when r47 was searched against protein conserved domain database maintained by ncbi , a trypsin - like ( 77 . 7 % of the conserved domain region ( 217 residues ) was aligned ) and pdz domain ( 73 . 3 % of the conserved domain region ( 86 residues ) was aligned ) were identified . these two domains were also associate with different serine proteases ( fig6 ). assessment of homologous protection induced by recombinant 56 kda protein from orientia tsutsugamushi in combination with 47 kda scrub typhus antigens in order to assess the protective effect of r56 and r47 proteins against infection , mice will receive varying doses of r56 and r47 protein components from the karp strain . since the immunization with 25 ug or 2 ug of r56 in mice showed a protective role in our previous experiments ( annual report j2 — 00_nmrc ), we will use the same doses for testing r56 in the present study . for the r47 proteins , we do not know the optimum doses , but we will start with two dosages of 5 ug and 25 ug at this time . alum and cpg will be used as adjuvants at the ratio of protein : al + 3 : cpg = 1 : 25 : 10 . alum has been approved for human use for a long time . vaccines prepared by antigens adsorbed onto alhydrogel have several advantages . the antigenic content is easier to standardize and the vaccine has good stability . alhydogel adsorbed antigens are simple and more convenient to use . in addition , the degree of local reaction following immunization is reduced . cpg induces a th1 - like pattern of cytokine production dominated by il - 12 and ifn - gamma with little secretion of th2 cytokines . female cd1 mice , 4 to 5 weeks old , 13 to 15 grams in weight , will be purchased from charles river laboratories ( wilmington , mass .). each study includes 5 - 7 groups of mice . a minimum of 15 - 20 mice is needed to down select the 20 % difference in protective efficacy at 80 - 85 % confidence level . each group of 20 mice will be immunized i . m . with the 56 kda antigen alone or in combination with r47 antigen except for the negative control groups . 15 mice will be challenged 4 weeks later and 5 mice will be sacrificed for measuring t - cell immune responses right before challenge . ( a ) antibody detection : mouse sera will be drawn immediately before immunization and before challenge . each serum sample will be assayed in duplicate by enzyme - linked immunosorbent assay ( elisa ) for antibodies against each antigen . anti - mouse igg and igm peroxidase - labeled conjugate ( boehringer - mannheim , indianapolis , ind .) will be tested . ( b ) cytokine detection in serum : cytokines have been studied physiologically and pathologically with a focus on the roles in immune regulation during inflammation . ifn gamma and il - 4 are cytokines for th1 and th2 cells , respectively . for many intracellular infections , il - 12 may induce th1 response , i . e . ifn - gamma production . the levels of ifn gamma and il - 2 were elevated in spleen mononuclear cells from 56 kda immunized mice . increased ifn was also detected in spleen cells and lymph nodes cells from mice chronically infected with orientia . it was noted that orientia was susceptible to inf gamma in cultured mouse cells . in the sera of patients with scrub typhus infection , the levels of g - csf , m - csf , ifn gamma , and tnf alpha were up regulated . mice blood will be drawn in heparin before immunization , 2 , 4 , 6 , 8 , 10 , 15 , and 30 days after immunization . sera samples will be assayed in duplicate for cytokine levels of ifn gamma , tnf alpha , il - 4 , il - 12 , g - csf , and m - csf . ( c ) cytokine production by antigen specific t - cells : spleen mononuclear cells ( smnc ), stimulated by recombinant antigens , will be monitored for the productions of ifn gamma and il - 2 at day 1 , 2 , and 3 post stimulation . the cells will be isolated by ficoll - hypaque ( pharmacia , uppsala , sweden ) density gradient centrifugation . after washing , the cells will be resuspended to a concentration of 5 × 10e6 per ml in warm rpmi - 10 , and 100 ul will be distributed in each well of a 96 - well plate as described . following incubation with corresponding recombinant antigens , culture supernatants will be collected and used in cytokine assays . the supernatants from triplicate smnc cultures will be pooled , and all samples will be assayed in triplicate . il - 2 activity in the supernatants from antigen - stimulated smnc will be tested for its ability to support growth of il - 2 - dependent ctll - 2 cells in the presence of anti - il - 4 antibody ( 11b11 ; 10 ug / ml ). for inf - gamma and il - 4 , the levels will be measured by enzyme - linked immunosorbent assay kit ( genzyme , boston , mass .) according to the manufacturer &# 39 ; s instructions . cd4 and cd8 cells will be selected to determine which cells are producing the cytokines using macs direct microbeads ( miltenyi biotec , auburn calif .). ( d ) proliferative response of spleen cells : smnc will be prepared as described above . after the stimulation by different amounts of recombinant protein or orientia karp , the cultures will be incubated at 37 c for 72 h under a humidified atmosphere of 5 % co2 . a total of 1 uci of [ 3h ] thymidine will be added to each well for the another 18 h of culture . cells will be harvested onto glass fiber filter strips , and incorporation of [ 3h ] thymidine will be counted in a liquid scintillation counter . prophetic examples for the treatment of hiv patient to reduce the virus load with immunoglobulins from subjects immunized by the immunogenic composition . there is no animal model for hiv infection at the present time . however , transgenic mice carrying the human cd - 4 gene can be infected by hiv . this type of mice will be used in the following experiments : mice will be infected with hiv . two months later , one group of mice will be treated with hyper immune plasma from the same type of mice immunized by the said immunogenic composition ( group 1 ). another group of mice will be treated with normal plasma to serve as the control group ( group 2 ). blood will be drawn from both groups at one week interval for four times . the hiv load in the blood will be quantitated by real time pcr . results from group 1 will be compared to group 2 to demonstrate the dereased virus load by the hyper immune plasma . the truncated 47 - kda protein was successfully cloned and overexpressed in bl21 cells . the over expressed protein was not in the inclusion body making the purification procedure complicated . the addition of detergent increased the amount of r47 suggesting the association of protein with membrane fraction . although dialysis of crude extract with 25 , 000 mw cutoff offered some purification and further purification using ph 8 . 0 or ph 7 . 0 buffer and salt gradient on an anion exchange column resulted in further purification , the protein was not yet fully purified . the pi of r47 was predicted to be 8 . 2 and suggested a cation exchange is more suitable for purification . experiments will continue to purify r47 with cation exchange followed by gel filtration to achieve desired purity . the inventive subject matter being thus described , it will be obvious that the same may be varied in many ways . such variations are not to be regarded as a departure from the spirit and scope of the inventive subject matter , and all such modifications are intended to be within the scope of the following claims .