AU767820B2 - HIV suppressor factor derived from scrub typhus - Google Patents

Description

WO 00/11035 PCT/US99/16725 HIV SUPPRESSOR FACTOR DERIVED FROM SCRUB TYPHUS BACKGROUND OF THE INVENTION 1. Field of the Invention The invention relates to a suppressor factor for HIV derived from mammals infected with Orientia tsutsugamushi (scrub typhus), and to compositions containing the suppressor factor and derivatives thereof for prevention and treatment of HIV infection.

2. Background Information Human Acquired Immune Deficiency Syndrome (AIDS) and infection by human immunodeficiency virus (HIV), its causative agent, have become serious worldwide problems since their emergence in the last several decades. In 1998, the United Nations estimated that 5.8 million individuals become newly infected with HIV each year The problem is particularly severe in developing countries. At present, there are no completely effective treatments for HIV infection or AIDS, although progress has been made in the development of antiretroviral drugs and drug combinations, which in some individuals have been effective in slowing the progress of the disease Antiretroviral drugs are expensive and are often poorly tolerated. Furthermore, greater than 90% of those currently infected with the HIV-1 virus will never have access to effective antiretroviral drugs Three or four antiretroviral drugs are often recommended both for treatment and also for prevention (post-exposure chemoprophylaxis) of HIV after a suspected high risk exposure Therefore, a need exists for a safer and more economical method of treatment and chemoprophylaxis.

0. tsutsugamushi is a small gram-negative bacillus that is distinct from other bacteria because of its obligate intracellular parasitism. It typically ranges in size from to 2.0 mm by 0.3 mm, but pleomorphism is common. 0. tsutsugamushi infection causes the disease called scrub typhus. The natural cycle of infection involves mites and small rodents. Humans are accidental hosts and acquire scrub typhus from the bite WO 00/11035 PCT/US99/16725 of an infected chigger. Neither direct mouse to mouse nor mouse to human transmission has been reported. Multiple serotypes of 0. tsutsugamushi cause illness and produce effective homologous immunity but transient cross-protection.

Signs and symptoms of scrub typhus vary from those of a mild, self-limiting illness to a fatal syndrome with failure of one or several major organ systems. An eschar at the site of the chigger bite is the most characteristic sign, but is not always present or goes unnoticed by the patient and physician. Direct injection of patient blood into mice remains the most sensitive method of isolating the causative organism, despite careful attempts at finding alternative methods.

The present inventors have discovered that AIDS patients who become infected by some, but not all, strains of scrub typhus experience a marked reduction in HIV viral load, with the maximum inhibitory effect at 0-14 days after admission to hospital. Sera obtained from some HIV negative patients with acute scrub typhus reduces HIV viral infectivity in vitro. Some scrub typhus sera reduce infectivity even more than HIV antibody positive sera in vitro. The interaction between scrub typhus and HIV appears to be bidirectional. Scrub typhus is no more severe in HIV seropositive individuals, but it is more difficult to isolate 0. tsutsugamushi from their blood (14% vs. 49% in HIV seronegative patients, Ref. 4).

SUMMARY OF THE INVENTION This invention takes advantage of the anti-HIV effects of an inhibitory factor produced during scrub typhus infection. The present inventors have discovered that scrub typhus infection reduces HIV viral load in patients who are also infected with HIV-1. In addition, sera from scrub typhus patients who are HIV seronegative exhibit anti-HIV- I activity in vitro. Various scrub typhus strains were cultivated and tested to study this phenomenon further. Sera from mice infected with strains which exhibited such activity are strongly reactive with HIV-1 p17 antigen peptides.

It is an object of the invention to provide a method of inducing an immunogenic response to human immunodeficiency virus (HIV), in particular to HIV-1. This immunogenic response is induced by exploiting the fact that certain epitopes present in scrub typhus are identical or similar to those present in HIV. The invention includes inhibitory factors, antibodies, antigens, peptides, immunogenic compositions and vaccines useful for prophylaxis and/or treatment of HIV infection, as well as methods for their use. After a scrub typhus strain with anti-HIV activity has been identified, WO 00/11035 PCT/US99/16725 isolation and testing of peptides from the organisms, purification of plasma and serum, and isolation of antibody from blood to be used in the compositions and methods of the invention can be carried out by routine methods known to those of skill in the art.

Accordingly, it is an object of the invention to provide a novel suppressor factor derived from scrub typhus infection (Orientia tsutsugamushi) which is useful for the prevention and treatment of HIV infection and AIDS.

In particular, it is an object of the invention to provide a suppressor factor derived from scrub typhus infection in the form of purified sera, plasma or immunoglobulin which is suitable for administration to patients at risk for, or infected with, HIV, either alone or in combination with other agents. It is a particular object of the invention to provide an isolated suppressor factor from sera or plasma taken from patients with scrub typhus which has anti-HIV activity. By "anti-HIV" activity is meant an effect of inhibiting HIV replication in vitro, or of reducing HIV viral load in vivo. Such inhibition or reduction is considered to occur if replication or viral load is reduced by at least about 3-fold, and more preferably by more than Such serum may be that obtained from an individual patient, or may be pooled from two or more patients.

As used herein, the term "isolated" is intended to mean separated from its natural environment. The term "purified" is intended to mean having naturally associated substances further removed, but not necessarily to 100% purity.

Purified sera, plasma or immunoglobulin which contains the anti-HIV suppressor factor according to the invention can be obtained by identifying patients having the suppressor factor according to the methods set forth hereinbelow and isolating and purifying the sera, plasma or immunoglobulin according to suitable methods known in the art.

It is also an object of the invention to provide peptides from inhibitory strains of scrub typhus. Such peptides function as antigens and can be used to produce antibodies effective for the prophylaxis and treatment of HIV infection. After isolation of inhibitory strains of scrub typhus, peptides useful for the invention can be made by routine methods which are familiar to those of skill in the art. The term "antigen", as used herein, includes such peptides, and particularly includes, inter alia, peptides from 6 to 100 and more preferably 12 to 72 amino acid residues in length which are capable of eliciting production of such antibodies. Such peptides can be made synthetically, for example, and screened for activity.

WO 00/11035 PCT/US99/16725 The term "antibody", as used herein, includes, but is not limited to, polyclonal antibodies as well as monoclonal antibodies, which may be naturally derived from any animal, synthesized in bacteria, or chemically or genetically synthesized. The antibody may be a whole antibody or an antigen binding fragment thereof and may in general belong to any immunoglobulin class. Thus, for example, it may be an immunoglobulin M antibody or an immunoglobulin G antibody. It is a particular object of the invention to provide an IgM and/or IgG anti-scrub typhus antibody that recognizes HIV p17 antigen or p24 antigen. By "anti-scrub typhus antibody" is meant antibody which recognizes, binds to, and/or inhibits the action of scrub typhus organisms. Serum, plasma or antibodies can be tested to determine whether it contains antibodies recognizing HIV p17 antigen or p24 antigen by means known to those of skill in the art. Such recognition can be demonstrated by immunoblotting techniques and/or enzyme-linked immunoassay (ELISA), as set forth hereinbelow, or by other standard techniques known to those of skill in the art.

It is a further object of the invention to provide scrub typhus antibodies which cross-react with HIV-1 proteins. By "cross-react with HIV proteins" is meant that the antibodies recognize and/or bind to HIV proteins.

It is a further object of the invention to provide an antibody that inhibits HIV virus replication by binding directly to HIV or to HIV infected cells and affecting any or all of the steps involved in the viral replication cycle.

It is a further object of the invention to provide a factor that suppresses HIV-1 virus replication. Such factor may be, for example, an antibody to scrub typhus which operates directly by binding to HIV, or otherwise by interfering in the replication process, or an antigen which stimulates production of such an antibody or antibodies.

The peptides of the invention may be used to produce monoclonal antibodies using, for example, hybridoma technology. These monoclonal antibodies can be administered to HIV infected patients as treatment and/or injected at intervals to HIV seronegative individuals as a means for preventing HIV infection (passive immunization). The peptides could also be administered to HIV negative individuals in order to stimulate production of anti-HIV antibodies (active immunization). Thus, the invention is contemplated to include compositions containing such peptides, including, but not limited to, vaccines, and methods for their use in prevention and treatment of HIV infection.

WO 00/11035 PCT/US99/16725 The compositions of the invention are also contemplated to be used to prevent maternal-fetal perinatal transmission of HIV.

As used herein, the term "vaccine" intended to mean both prophylactic and therapeutic immunogenic compositions for prevention and treatment of HIV infection.

Such immunogenic compositions may be active, containing an effective amount of a peptide according to the invention which is capable of eliciting in a host a protective immune response to HIV infection, along with a pharmaceutically acceptable diluent or carrier. Alternatively, such immunogenic compositions may be used to generate antibodies according to the invention which can be administered in an amount which is sufficient to confer a protective or therapeutic effect.

The immunogenic compositions of the invention are administered in amounts readily determined by persons of ordinary skill in the art by routine experimentation.

In vaccines, and when used otherwise for prophylaxis or therapy, the immunogenic compositions of the present invention may be used in conjunction with pharmaceutically acceptable adjuvants, carriers, diluents and excipients. In particular, the immunogenic composition may further comprise at least one adjuvant, such as aluminum phosphate or aluminum hydroxide. Suitable advujants, excipients, diluents or carriers will be known by those skilled in the art, and can be found in, for example, Remington's Pharmaceutical Sciences 18 h ed., 1990).

The preferred means of administration compositions of the invention which are contemplated to be used for immunization and/or treatment is via intramuscular, intravenous or subdermal injection. However, they may be administered by any conventional means that are determined by those of skill in the art to be safe and effective, including oral and mucosal delivery.

The invention differs from previous approaches to HIV therapy and prevention because it does not rely on working with HIV virus. Instead, it relies on the novel, cross-reactive inhibitory effects bestowed by an entirely different organism.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (upper panel). HIV RNA copy numbers (expressed as percent of the day 28 measurement) are shown for the 10 individuals with scrub typhus (red circles) and the patients with acute non-typhus infections (blue circles). The median viral loads (solid squares) of scrub typhus patients are connected by a red line and those of the control group by a blue line.

WO 00/11035 PCT/US99/16725 Figure 1 (lower panel). Sequential HIV RNA concentration in plasma of the 4 patients coinfected with HIV and scrub typhus who had at least a 3-fold reduction in copy number relative to the day 28 value. RNA copy numbers were below the limits of detection in the 2 patients displayed in red; ST2 (day 14) and ST3 (days 0,3 and 7).

Viral load was assayed by Roche Amplicor RT-PCR in one patient (blue dotted line) and by a bDNA assay in the others.

Figure 2. IFA performed with HIV-1 infected lymphocytes and sera taken 1 month after mice were infected with scrub typhus. The polar staining (top left X40) and bottom (X100) is not seen with preimmune mouse sera (top right Figure 3. ELISA data at day 0 (presentation with scrub typhus), day 14 and day 600 post presentation for a scrub typhus patient who is not infected with HIV. P13, P41 and P105 are peptides derived from HIV P17 gag protein. The blue shows IgM reactivity and the red shows IgG reactivity. The data demonstrate that an HIV uninfected individual gains immune reactivity to epitopes on HIV proteins during scrub typhus infection.

Figure 4. Scrub typhus infection causes mice to acquire stong antibodies to a gag p17 epitope. The response at day 28 is IgM antibody. SV40 is one strain of scrub typhus.

Figure 5. Reactivity to peptide 41 in groups of mice infected with 3 different strains of scrub typhus (240, 155, krp) and a control group. The serum reactivity of each group is compared to a control peptide from a different region of p17 (pep 129) DETAILED DESCRIPTION OF THE INVENTION Informed consent is obtained from human subjects who receive full counseling for HIV testing under a protocol approved by the participating institutions located at Chiangrai Regional Hospital and Maesod Hospital (Ministry of Public Health, Thailand) and at the Armed Forces Research Institute of Medical Sciences, Bangkok (AFRIMS). Animal research is conducted in compliance with the Animal Welfare Act and animal use protocols were approved by the AFRIMS Institutional Animal Care and Use Committee.

WO 00/11035 PCT/US99/16725 Example 1 Scrub typhus infection reduces HIV-1 viral load HIV-1 viral load was measured in patients infected with HIV-1 who were also diagnosed as having scrub typhus. The study was carried out as follows.

Scrub typhus was initially diagnosed in adults with febrile illnesses of 1 week's duration or less and appropriate clinical features by dot-blot immunoassay and confirmed by an indirect immunoperoxidase assay. Leptospirosis was diagnosed by an IgM specific ELISA (Panbio, Brisbane, Australia) of proven specificity in Thailand Watt; unpublished data). HIV-1 antibody was detected using two rapid screening tests (SerodiaR-HIV, Fujirebio Inc., Tokyo, Japan, and CapillusR HIV-1/HIV-2, Cambridge Diagnostics, Galway, Ireland). Confirmatory HIV-1 Western blot (NovapathR HIV-1 Immunoblot, Bio-Rad, Hercules, California, USA) was performed if a screening test result was unclear or if there was a discrepancy between the results of 2 screening tests, in accordance with Thai government guidelines.

HIV-1 RNA levels were measured in EDTA plasma using commercial kits (RT-PCR, Amplicor HIV Monitor test; Roche Molecular Systems, Branchburg, NJ,- limit of detection 50 copies/ml, CV 24%; and Chiron 2 nd generation bDNA assay, Chiron Corporation, Emeryville, CA- limit of detection 500 copies/ml, CV The same method was used for longitudinal assessment. CD4 counts were monitored using 2color flow cytometry and a FACScan instrument (Becton Dickinson Immunocytometry Systems, San Jose, CA).

HIV-1 subtyping and isolation HIV subtype was determined using peripheral blood mononuclear cells (PBMCs) from infected subjects and nested PCR employing primers which differentiate HIV subtypes B and E. PBMCs from infected subjects were cocultured with phytohemagglutininstimulated PBMCs from a seronegative Thai donor at a 1:1 ratio. The biological phenotype was determined on admission HIV isolates obtained from patients with <200 CD4+ cells. Supernatants from positive virus cultures were inoculated onto the CD4+ T cell line MT-2 using the technique described by the Division of AIDS, NIAID Cultures were scored as either syncytium inducing (SI) or non-syncytium inducing

(NSI).

WO 00/11035 PCT/US99/16725 Follow-up of patients HIV seropositive subjects were evaluated clinically and by HIV viral loads 3, 7, 14 and 28 days after admission to the study. Patients were prospectively excluded from viral load analysis if there was clinical or laboratory suspicion of opportunistic or other infections, without knowledge of HIV viral load. PCR for HIV DNA was carried out on samples where viral load was below the limit of detection using primers to clade B and E viruses. Patients were treated with either chloramphenicol or doxycycline for scrub typhus, quinine or chloroquine for malaria, and penicillin for leptospirosis. The fever clearance time (FCT) was the number of hours between the first dose of medication and the first time when the morning oral temperature was 37.2 0 C in the absence of antipyretic medication. Patients infected by HIV-1 were treated with the highest practically attainable standard of care in Thailand, which during the duration of this study did not include specific antiretroviral agents.

An estimated 38,200 fever patients were screened during 12 study months at Chiangrai; fewer at Maesod. Scrub typhus was diagnosed in 173 patients; 22 of whom were known to be HIV seropositive. HIV serotesting identified an additional 19 subjects for inclusion. Thirty-one of these 41 patients were excluded from the final viral load analysis because additional pathogens were identified in blood or cerebrospinal fluid after enrollment because clinical or laboratory findings were atypical of scrub typhus, malaria or leptospirosis and because fever cleared initially but then recurred prior to day 28 Longitudinal viral load measurements from 15 patients were included in the final analysis (Figure Nine patients infected with 0. tsutsugamushi, 1 with leptospirosis, and 1 with P. falciparum were studied in Chiangrai. Two patients with falciparum malaria, and one each with vivax malaria and scrub typhus were followed in Maesod.

Only 4 of these 15 patients were previously known to be HIV seropositive.

Admission characteristics of the 10 patients infected with 0. tsutsugamushi were generally similar to admission characteristics of the 5 non-typhus infected individuals (Table 1).

WO 00/11035 PCT/US99/16725 Table 1. Admission characteristics of the 15 HIV-1 infected subjects whose viral load measurements were included in the final analysis. The standard deviation is shown in parentheses for mean values, the range for median values.

Scrub typhus malaria(4) leptospirosis(l) Demographic Information Age Male/female Thai/hilltribe 31(±11) 6/4 Non-typhus 41(+14) 3/2 2/3 Chiangrai/Maesod Clinical Days of fever Oral temperature Hematocrit White blood cells/mm 3 Platelets (X 1,000)/mm 3 CRP (mg/L) CD4 cells/mm 3 HIV RNA (copies/ml) 958,513) 4(1-7) 38.2 0 C(+0.7) 34%(+6) 5,915(1,420-27,390) 120(63-357) 40.9(0-146.2) 117(8-403) 59,220(0*-2,484,677) 3 (3-7) 38.2 0 C(+0.7) 34%(+5) 7,260(5,720-7,900) 63(40-94)* 96.5(38.8-169.6) 255(153-299) 384,740(10,000p< 0 0 0 below limit of detection of assay WO 00/11035 PCT/US99/16725 Platelet counts were significantly higher (p<0.05) in scrub typhus patients. CD4 cell counts and HIV-1 RNA copy numbers were lower, but not significantly so in 0. tsutsugamushi-infected individuals. The median FCT in 0. tsutsugamushi infection was 64h (range 41h-72h) and 66h (range 17h-67h) in the non-typhus group.

The median viral load 3 days after admission was significantly lower in the scrub typhus group (193% vs 376%, p<0.05) as was the pooled median value for all loads measured from days 0-14 (108% vs 298%, p<0.05), compared with the non-typhus group. HIV RNA copy number fell below the threshold of detection in 2 individuals infected with 0. tsutsugamushi, and 2 PBMC samples were also HIV DNA negative by PCR (Figure Relative to the day 28 measurement, there was a 3-fold or greater reduction of viral load in 4 of the 10 patients with acute scrub typhus (2 received doxycycline and 2 received chloramphenicol) but in none of the non-typhus infected individuals (p=0.15).

CD4+ lymphocytes were quantified 1 month after admission in 11 patients 3 with a fall in viral load, 8 without. One month specimens were not collected from the other 4 subjects. CD4 cell counts were higher at 1 month than on admission by 166%, 174% and 52% in the 3 patients with a fall in copy number but fell by a median of 16% (range -42% to in the other 8 individuals (p Primary HIV-1 viral isolates were obtained on admission from 17 patients with CD4 counts of 200 cells/mm 3 6 of whom were included in the final viral load analysis (Table 2, numbers 1-6).

WO 00/11035 PCT/US99/16725 Table 2. Phenotype of admission HIV-1 viral isolates (labelled 1-17), classified as either syncytia inducing (SI) or non-syncytia inducing (NSI).

Phenotype CD4 cells/ml Scrub typhus 1. NSI 2. NSI 118 3. NSI 9 NSI 161 NSI 116 6. NSI 66 7. NSI 107 8. NSI 7 9. NSI 131 NSI 147 Non-typhus 11. SI 17 12. SI 31 13. NSI 9 14. SI SI 146 16. SI 22 17. NSI 175 subtype B, all others were subtype E.

Five of the 7 isolates from non-typhus infected individuals were SI variants, but all admission isolates obtained from 0. tsutsugamushi-infected patients were of the NSI phenotype A single isolate Table 2) was HIV-1 subtype B, the remainder were subtype E virus.

Acute phase reactants and immune activation.

TNF-alpha was undetectable in admission sera from all 8 0. tsutsugamushiinfected patients tested (no specimens were available from 2 patients) and from 2 of non-typhus infected individuals; concentrations were 24.0, 58.4 and 61.5 pg/ml in the other 3. TNF concentrations on day 28 were below detectable limits in all 8 scrub WO 00/11035 PCT/US99/16725 typhus and all 5 non-typhus specimens tested. There were no significant differences between median admission CRP concentrations in patients with scrub typhus (40.9 mg/L, range 0-146.2) and those without (96.5 mg/L, range 38.8-169.6; p> 0 Day 28 CRP concentrations were below the limit of detection (5mg/L) in the 8 sera from the scrub typhus group and in 3 of 5 sera from non-typhus infected patients. Day 28 CRP concentrations in the other 2 malaria-infected subjects were 7.7 and 6.3 mg/L; CRP concentrations exceeding >8.0 mg/L have been considered elevated in previous malaria studies Macrophage inflamatory protein-1 alpha (MIP-1 alpha) concentrations were below detectable limits and RANTES measurements were lower than the median reference value (49,137 pg/ml) and were within or below normal range in all 12 specimens assayed. RANTES concentrations did not correlate with viral load 0.26; p=.43).

Plasma HIV-1 viral load fell by 3-fold or more in 4 of 10 patients not receiving antiretroviral therapy during acute scrub typhus infection (Figure Four measurements were below the limits of detection and in 2 instances HIV-1 DNA could not be detected by nested PCR (Figure 1, lower panel). No infectious agent has previously been demonstrated to reduce HIV viremia in AIDS patients. Rather, median plasma RNA copy numbers increased 7.8 fold during a prospective study of acute illness caused by a variety of pathogens (10) and transient elevations of viral load have been reported with intercurrent infection, immunization and even tuberculin skin testing (11-14). The RNA copy number 3 days after enrollment was significantly lower in 0. tsutsugamushi-infected individuals than in the non-typhus group (p<0.05) even though a marked drop in viral load occurred in only a subset of scrub typhus patients. This suggested that inter-patient variability in HIV-1 suppression results from serologically unique epitopes of individual strains of 0. tsutsugamushi There was a significant association between scrub typhus and non-syncytiainducing HIV variants (Table 2, p <0.001). NSI viruses are associated with delayed disease progression and slower viral replication Scrub typhus may restrict HIVinduced syncytia formation by influencing p17 protein, which is found in the membrane of HIV-infected lymphocytes and facilitates cell to cell spread of HIVvirus Sera obtained during acute 0. tsutsugamushi infection had potent HIVneutralizing activity and was reactive with p17 recombinant protein (Table 3).

WO 00/11035 PCT/US99/16725 Regardless of mechanism, it is clear that and agent produced as a consequence of scrub typhus infection is effective in inducing immunogenicity to HIV.

Table 3. Human (KDX5) and mouse (SV240) immunoassays. "ND" indicates not done.

a) Reduction of infectivity p24 antigen compared to normal human sera) PBMCs Day 0 Day 28 HIV pos 96h incubation 70% 48% -27% 120h incubation 73% 73% 2% H9 cells whole sera 92% 48% 100% antibody-depleted sera 42% 3% 66% b) p17 ELISA serum dilution 1:200 1:400 Day 14 0.53 0.32 Day 450 0.07 0.05 HIV pos 0.27 0.14 c) IgM peptide scanning (Sigma units) amino acids Day 0 Day 14 Day 28 Day 450 HIV pos 13-24 105-116 109-120 189.9 163.1 200.2 82.3 172.1 179.3 0 252.6 1.1 SV240 a) Syncytia per well (median 95%C.I.) Experiment 1 Experiment 2 b) IgM p 7 Western blot reactivity Pre-immune 101 (98-105) 145 (140-161) Day 28 48 (42-56) 76 (61-78)

YES

WO 00/11035 PCT/US99/16725 Peptide scanning of sera obtained early in infection revealed IgM reactivity at several regions within the p17 protein. Convalescent sera (day 450) neither reacted with recombinant p1 7 protein nor with synthetic p17 peptides. Immune serum from scrub typhus-infected mice was reactive with the p17 band on HIV Western blot, inhibited syncytia formation of HIV-infected lymphocytes, and fluoresced strongly by IFA.

The results demonstrate that acute infection with Orientia tsutsugamushi results in a marked reduction of the HV- 1 viral load in some AIDS patients.

Example 2 Anti-HIV activity of serum from scrub typhus patients Sera taken from scrub typhus patients who were HIV-1 seronegative was tested for effect on HIV-1 activity in vitro.

Syncytia inhibition by mouse sera. Syncytia were counted 4 days after CEM- SS cells were infected with a laboratory-adapted clade E HIV-I strain (NPO3) at 1:20 or 1:80 dilution of test sera Samples and preimmune controls were run in quadruplicate.

Infectivity reduction assays PHA-stimulated PBMCs from a US donor were inoculated for 1.5h with a primary, NSI HIV subtype E isolate (NP 1660). The inoculum was removed, and cells washed and incubated for 18-24h in media containing interleukin-2. Test sera were then added at a dilution of 1:20 with normal human sera (NHS) and sera from HIV positive donors as controls. Similar experiments were performed using H9 cells and NPO3 virus before and after protein G antibody depletion. The inhibition was 1 (p24 test sample/ p24 NHS) X 100.

Immunoassays (Table Sera from 1 HIV-uninfected scrub typhus patient (coded KDX5) had neutralizing activity exceeding that of HIV positive control sera and was further characterized. KDX5 HIV-inhibition was markedly reduced after antibody depletion. There was IgG ELISA reactivity to p17 recombinant protein in sera taken 14 days after scrub typhus infection, but not in day 450 sera. Day O and day 14 sera (but not day 28 or day 450 sera) had IgM reactivity to several p17 epitopes by peptide scanning, including an epitope reactive with HIV positive serum. Convalescent sera from mice infected with scrub typhus (isolate SV240) inhibited HIV syncytia formation and was further tested. Day 28 SV240 serum (but not preimmune serum) reacted strongly with the p17 band and weakly with the p55 band on IgM-specific Western blot WO 00/11035 PCT/US99/16725 and gave a fluorescent signal when incubated with HIV-infected lymphocytes (Figure 2).

The results demonstrate that sera taken from HIV-1 seronegative patients during and after infection with scrub typhus shows anti-HIV-1 activity in vitro.

Example 3 Cross-reactivity of scrub typhus antibodies with HIV-1 p 1 7 gag protein IgG and IgM antibodies made during and after an acute scrub typhus infection were tested for cross-reactivity with HIV-1 using standard ELISA techniques.

The results, shown in Figure 3, demonstrate that IgG and IgM antibodies made during acute scrub typhus infection are cross-reactive with HIV-1 p17 gag protein. Scrub typhus infection also causes mice to acquire strong antibodies to a gag p 7 epitope, as shown in Figure 4. Reactivity to p17 peptides (pep 41 and pep129) for mice infected with three different strains of scrub typhus is shown in Figure Example 4 Sequence similarity between surface antigen of Orientia tsutsugamushi and HIV-1 gag gene.

The sequences of surface antigen of 0. tsutsugamushi and HIV-1 gag gene were compared. The results are shown in Tables 5-7.

WO 00/11035 WO 0011035PCTIUS99/1 6725 Table SAMINO ACID MAXIMUM HOMOLOGY LIST PROS IS

<<INFORMATION>>

EQUIVALENT

1 Ala (A),Ser(S),Thr(T),Pro(P),Gly(G) 2 Asn(N), Asp(D), Asx(B),Glu(E),Gln(Q),Glx(Z) 3 His(H),Arg(R),Lys(K) 4 Met(M), Leu(L),Ile(I),Val(V) Phe(F), Tyr(Y),Trp(W) FILEl NAME START END FILE2 NAME START PROT.AMI 1 511 56KD.AMI1

END

8 FILE NAME PROT .AMI 56KD.AMI FILE NAME

PROT.AMI

56KD.AMI FILE NAME

PROT.AMI

56KD.AMI FILE NAME 56KD.AMI FILE NAME

PROT.AMI

56KD.AMI FILE NAME PROT .AMI 56KD.AMI 1 10 20 30 40 50 MGARASVLSGGELDRWEKIRLRPGGKKKYKLKH

IVWASRELERFAVNPGLLETSEGGRQI

-184 -175 -165 -155 -145 -135 -125 61 70 80 90 100 110 120 LGQLQPSLQTGSEELRSLYNTVATLYCVHQRI

EIKDTKEALDKIEEEQNKSKKKAQQAAA

-124 -115 -105 -95 -85 -75 121 130 140 150 160 170 180

DTGHSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGAT

-64 -55 -45 -35 -25 -15 181 190 200 210 220 230 240

PQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDKVMPVMAGPIAPGQMREPRGSDIAGTT

-4 5 15 25 35 45 241 250 260 270 280 290 300 STLQEQIGWMTNNPPIPVGEIYKRWI

ILGLNKIVRMYSPTSILDIRQGPKEPFRDYVRFY

56 65 75 85 95 105 115 301 310 320 330 340 350 360 KTLRAEQASQEVKNWMTETLLVQNANPDCKTI

LKALGPAATLEEMMTACQGVGGPGHKAR

116 125 135 145 155 165 175 WO 00/11035 WO 0011035PCTIUS99/1 6725 FILE NAME 56KD.AMI FILE NAME

PROT.AMI

56 KD.AMI 361 370 380 390 400 410 420 VLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGH IARNCRAPRKKGCWKCGKEGH 176 185 195 205 215 225 235 421 430 440 450 460 470 480

QMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPFLQSRPEPTAPPEESFRSGVET

236 245 255 265 275 285 295 FILE NAME

PROT.AMI

56KD.AMI 481 490 500 510 TTPSQKQEPIDKELYPLTrSLRSLFGNDPSSQ 296 305 315 325 MATCHING PERCENTAGE TOTAL WINDOW 1% ALIGNMENT WINDOW 80% 511) WO 00/11035 WO 0011035PCTIUS99/1 6725 Table 6 PROT .AMI STA56 .AMI

PROT.AMI

STASE .AMI PROT .AMI STA56 .AMI PROT .AMI STA56 .AMI

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STA56 .AMI

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STA56 .AMI 20 30 40 50 MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGGRQ

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SAMSALSLPFSASAI ELGEEGLECGPYAKVGVVGGMITGVESARLDPADAEGKKHLSLTN 20 30 40 50 80 90 100 110 120 LGQLQPSLQTGSEELRSLYNTVATLYCVHQRI EIKDTKEALDKI EEEQNKSKKKAQQAAA

GLPFGGTLAAGMTIAPGERAEIGVMYLTNITAQVEEGKVKADSVGETKADSVGGKDAPIR

80 90 100 110 120 130 140 150 160 170 180 DTGHS SQVSQNYP IVQNIQGQMVHQAI SPRTLNAWVKVVEEKAFS PEVI PMFSALSEGAT KRFKLTPPQPTIMPISIAVRDFGIDI PNQTSAASTSRSLRLNDEQRAAARIAWLKNCAGI 130 140 150 160 170 180 190 200 210 220 230 240 PQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDKVMPVMAGP

IAPGQMREPRGSDIAGTT

DYRVKNPNDPNGPMfVINPILLNI PQGNPNPVGNPPQRANPPAGFAIHNHEQWRHLVVGLA 190 200 210 220 230 240 250 260 270 280 290 300 STLQEQIGWM4TNNPPI PVGEIYKRWI ILGLNKIXTRMYSPTS ILDIRQGPKEPFRDYVRFY V V

ALSNANKPSASPVKVLSDKITQIYSDIKHLADIAGIDVPDTSLPNSASVEQIQNKMQELN

250 260 270 280 290 300 310 320 330 340 350 360

KTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKAR

DLLEELRESFDGYLGGNAFANQIQLNFVMPQQAQQQGQGQQQQAQATAQEAVAAAAVRLL

310 320 330 340 350 360 370 380 390 400 410 420 VLAEAMSQVTNSAT IMMQRGNFRNQRKIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGH NGNDQ IAQLYKDLVKLQRHAG IKKAMEKLAAQQEEDAKNQGEGDCKQQQGTSEKSKKGKD 370 380 390 400 410 420 430 440 450 460 470 480

QMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPFLQSRPEPTAPPEESFRSGVET

KEAEFDLSMIVGQVKLYADVMITESVS IYAGVGAGLAYTSGKIDNKDI KGHTGMVASGAL 430 440 450 460 470 480 490 500 510

TTPSQKQEPIDKELYPLTSLRSLFGNDPSSQ

GVAINAAEGVYVDI EGSYMYSFSKIEEKYSI 490 500 510 WO 00/11035 WO 0011035PCT/US99/1 6725 Table 7 PROT.AMI MGARASVLSGGELDRWEKIRLRPGGKKKYKLKH IVWASRELERFAVNPGLLETSEGGRQI STA56 .AMI PROT .AMI STA56 .AMI

PROT.AMI

STA56 .AMI

PROT.AMI

STA56 .AMI

PROT.AMI

STA56 .AMI

PROT.AMI

STA56 .AMI

PROT.AMI

STA56 .AMI

ADSVGGKDAPIRKRFKLTPPQPTIMPISIAVRDFGIDIPNQTSAASTSRSLRLNDEQRAA

120 130 140 150 160 170 80 90 100 110 120 LGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEI KDTKEALDKI EEEQNKSKKKAQQAAA ARIAWLKNCAGIDYRVKNPNDPNGPMVINPILLNI PQGNPNPVGNPPQRANPPAGFAIHN 180 190 200 210 220 230 130 140 150 160 170 180 DTGHSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVI PMESALSEGAT HEQWRHLVVGLAALSNANKPSAS PVKVLSDKITQ IYSDIKH-LADIAGIDVPDTSLPNSAS 240 250 260 270 280 290 190 200 210 220 230 PQDLNTMLNTVGGHQAA14-QMLKETINEEAAEWDKVMPVMAGP IAPGOMREPRGSDIAGT v V VEQIQNKMQELNDLLEELRESFDGYLGGNAFANQ IQLNFVMPQQAQQQGQGQQQQAQATA 300 310 320 330 340 350 240 250 260 270 280 290 TSTLQEQIGWMTNNPPI PVGEIYKRWI ILGLNKIVRNYSPTSILDIRQGPKEPFRDYVRF QEAVAAAAVRLLNGND- QIAQLYKDLVKLQRH-AG I KKAEKLAAQQEEDAKNQGEGDCKQ 360 370 380 390 400 410 300 310 320 330 340 350

YKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKA

QQGTSEKSKKGKDKEAEFDLSMIVGQVKLYADVMI TESVSIYAGVGAGLAYTSGKIDNKD 420 430 440 450 460 470 360 370 380 390 400 410

RVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHIARNCRAPRKKGCWKCGKE

IKGHTGMVASGALGVAINAAEGVYVDI EGSYMYSFSKI EEKYS INPLMASVSVRYNFQT 480 490 500 510 520 530 WO 00/11035 PCT/US99/16725 The results show that there are significant similarities between the amino acid sequences of an antigen on the surface of Orientia tsutsugamushi and sequences encoded by the HIV-1 gag gene.

Example Scrub typhus strains with anti-HIV activity can be identified as follows.

1. Isolation of Orientia tsutsugamushi from human blood 0.2 cc of whole blood from patients with acute scrub typhus is injected intraperitoneally (IP) into each of 3 mice using a sterile 26 gauge 1 inch needle. The needle is changed between mice and the abdomen cleansed with alcohol before each injection. Mice are checked daily for 14 days and euthanized if they show signs of illness. Spleens are harvested and divided into two sections. One half is placed in a vial and stored at -70 0 C, and the other is homogenized with 2.0 ml of Snyder's solution to make a 10-20% suspension.

Two or more mice are injected IP with 0.2 ml of the suspension and observed for 28 days. If they become sick rough coat, inactivity) within 28 days they are euthanized. Giemsa stained peritoneal scrapings are checked for the presence of 0. tsutsugamushi and the spleens from infected mice stored at -70 0 C, as detailed by Watt et al. Blood from about forty patients with scrub typhus should yield about 10 new isolates of the organism.

2. Immunoassays To generate immunological reagents, outbred ICR mice are infected with O.

tsutsugamushi (1000 X mouse lethal doses 0 intraperitoneally) using organisms isolated from patients with scrub typhus as previously described Sera are obtained 28 days after infection by intracardiac puncture. Sera are assayed on Western blots of whole HIV-1 viral lysate protein (NovapathR HIV-1 Immunoblot, Bio-Rad, Hercules, California, USA) using standard secondary antibodies labelled with horse-radish peroxidase and an anti-mouse IgM heavy chain conjugate. Positive and negative controls supplied by the manufacturer and preimmune sera are used in comparisons. Detection of HIV reactive antibodies is conducted using indirect immunofluorescence with H9 cells infected with HIV-1 (NPO3) for 96h. Test sera are incubated with the cells at 37°C for 30min, washed and incubated with FITC-labelled anti-IgG conjugate. Positive control antibody is mouse monoclonal anti-gag antibody produced against HIV subtype B (NIH AIDS Research and Reference Reagent Program).

Negative controls are uninfected H9 cells and pre-immune antisera.

WO 00/11035 PCT/US99/16725 Human sera are assayed by ELISA for p17 antibody using recombinant p17 HIV-1 core protein (Immunodiagnostics Inc, Bedford, MA) at a coating concentration of 1 ug/ml.

Positive controls are sera from HIV-infected subjects reactive with p17 antigen by Western blot. Negative controls included sera from HIV-uninfected individuals and sera added to wells not coated with p 7 antigen. Eight amino acid (aa) overlapping 12-mer peptides were synthesized according to the p17 aa sequence of a Northern Thai HIV clade E isolate (CM243). For results presented herein, ELISA reactivities were assayed as previously described except that an IgM-specific conjugate was used. Experiments included sera from HIV infected patients as well as HIV uninfected patients with acute scrub typhus.

Acute phase reactants and immune activation.

Serum C-reactive protein (CRP) concentration was measured by serial radial diffusion with a lower limit of detection of 3.3 mg/L; values above 5.0 mg/L were abnormal. TNF-alpha concentration was determined by ELISA using commercially available kits (R&D Systems, Minneapolis, Minn) with an assay range of 15.6-1,000 pg/ml. RANTES (normal range 24,230-85,990 pg/ml) and MIP-1 alpha (limit of detection 80 pg/ml) were assayed by ELISA using commercially available kits (R&D Systems) on the first five HIV seropositive patients infected with 0. tsutsugamushi.

Statistical Analyses Repeated measures were analyzed using multivariate analysis and the Linear General Hypothesis (SYSTAT v5.2, SYSTAT Corp. Evanston, IL). Proportions were compared with chi-square analysis or Fisher's exact test (StatView, SAS Institute Inc. Cary, NC). Values not normally distributed were analyzed by the Mann-Whitney U statistic or Kolmogorov- Smirnoff test as appropriate. Two-sided tests of significance were measured in all cases.

Changes in viral loads were related to the measurement made 28 days after admission to the study in accordance with recommendations made by an NIH advisory panel This time point most closely represents viral status after resolution of symptoms and signs of acute infection and completion of therapy. Raw viral load data were converted to percentages related to the day 28 value (100%) to adjust for inter-assay differences. Changes greater than threefold in either direction on repeated measurements are considered to reflect a biologically relevant change in the level of plasma HIV RNA For peptide scanning, optical densities were expressed in standard deviations from the median reactivity to background peptides; reactivity more than 5 times that of background was considered significant.

P:\OPER\P.k\2388474-183clims.doc-2 1/o0803 -22- Useful variations of this method and other means for identifying such strains will be apparent to those of skill in the art.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form or suggestion that that prior art forms part of the common general knowledge in Australia.

P'AOPERUP,k388474-18kd.n- do l UI011 -22A References cited herein are listed for convenience and are hereby incorporated by reference.

REFERENCES

1. Bloom, BR. The highest attainable standard:ethical issues in AIDS vaccines. Science 1998;279: 186-188.

2. Egger, M, Hirschel, B, Francioli, P, et al. Imrpact of new antiretroviral combination therapies in HIV infected patients in Switzerland: prospective rnulticentre study. Brit.

Med J. 1997; 315:1194-9.

3. Katz, MH, Gerberding, IL. The care of persons with recent sexual exposure to M1V. Ann Intern Med 1998;128:306-12.

4. Kantipong, P. Watt, .ongsakul, K, Chuenchitra, C. HIV infection does not influence the clinical severity of scrub typhus. Guin. Inf. Dis. 1996: 23:1168-70.

Watt G, Strickrnan:D, Kantipong Jongsakul. K. Paxton H_ Prospective evaluation of a dotbiot immunoassay -for the rapid diagnosis of scrub typhus. J Inf Dis 1998; 177:800-2.

6. Winslow WET, Merry DJ, Pirc ML, Devine PL. Evaluation of a commercial enzymelinked immunosorbent assay for detection of immunoglobulin M antibody in diagnosis of human leptospiral infection. I Clin Micro 1997; 35:1938-42.

Artenstein AW, VanCott TC, Mascola. J et al.. Dual. infection with human immunodeficiency virus type I of distinct envelope subtypes in humans. J Inf Dis 1995; .:171:805-10.

WO 00/11035 PCT/US99/16725 8. Division of AIDS, National Institute of Alllergy and Infectious Diseases, 1997. DAIDS Virology Manual for HIV Laboratories. Publicaton NIH-97-3828. U.S. Department of Health and Human Services, Washington D.C.

9. McGuire W, D'Alessandro I, Olaley BO, Thomson MC, Langerock P, Greenwood BM, Kwiatowski D. C-reactive protein and haptoglobin in the evaluation of a communitybased malaria control program. Trans Roy Soc Trop Med Hyg 1996; 90:10-14.

Sulkowski MS, Chaisson RE, Karp CL et al. The effect of acute infectious illnesses on plasma human immunodeficiency virus (HIV) type 1 load and the expression of serologic markers of immune activation among HIV-infected adults. J Inf Dis 1998; 178:1642-8.

11. Mole L, Ripich S, Margolis D, Holiday M. The impact of active herpes simplex virus infection of human immunodeficiency virus load. J Inf Dis 1997; 176:766-70.

12. Brichacek B, Swindells, Janoff EN et al. Increased plasma human immunodeficiency virus type 1 burden following antigenic challenge with pneumococcal vaccine. J Inf Dis 1996; 174:1191-9.

13. Ortigao-de-Sampaio, Shattock RJ, Hayes P, Griffin GE, Linhares-de-Carvalho MI, Ponce de Leon A, Lewis DJM, Castello-Branco LRR. Increase in plasma viral load after oral cholera immunization of HIV-infected subjects. AIDS 1998; 12:F145-50.

14. Garcia F, Vidal G, Gatell JM et al. Changes in HIV-1 RNA viral load following tuberculin skin test. JAIDS 1998; 18:398-9.

Oaks EV, Rice RM, Kelly DJ, Stover CK. Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens. Inf Imm 1989; 57:3116-22.

16. Richmann DD, Bozzette SA. The impact of the syncytium-inducing phenotype of human immunodeficiency virus on disease progression. J InfDis 1994; 169:968-74.

17. Fais S, Capobianchi MR, Abbate I, Castilletti C, Gentile M, Fei PC, Ameglio F, Dianzani F. Unidirectional budding of HIV-1 at the site of cell to cell contact is associated with co- WO 00/11035 PCT/US99/16725 polarization of intercellular adhesion molecules and HIV-1 viral matrix protein. AIDS 1995; 9:329-35.

18. Nara PL, Fischinger PJ. Quantitative infectivity assay for HIV-1 and Nature 1988; 332:70.

19. Zolla-Pazner S, Alving C, Belshe R et al. Neutralization of a clade B primary isolate by sera from human immunodeficiency virus-uninfected recipients of candidate AIDS vaccines. J InfDis 1997; 175:764-74.

Watt G, Chouriyagune C, Ruangweerayud R et al. Scrub typhus infections poorly responsive to antibiotics in Northern Thailand. Lancet 1996; 348:86-89.

21. Loomis-Price LD, Cox JH, Mascola JR et al. Correlation between humoral responses to human immunodeficiency virus type 1 envelope and disease progression in early-stage infection. J Inf Dis 1998; 178:1306-16.

22. Report of the NIH panel to define principles of therapy of HIV infection. Ann Int Med 1998, 128:1057-78.

EDITORIAL NOTE APPLICATION NUMBER 56681/99 The following Sequence Listing pages 1/14 to 14/14 are part of the description. The claims pages follow on pages 25 to 26.

IlLin nn/l I n1c I h~ 'V A 1 14 r..Iu~ti SEQUENCE LISTING <110> WATT, GEORGE H DESOUZA, MARK S BROWN, ARTHUR E SIRIYAPHAN, PHINO BIRX, DEBORAH L <120> NOVEL HIV SUPPRESSOR FACTOR DERIVED FROM SCRUB TYPHUS <130> WATT <140> <141> /43 <150> <151> 60/097,288 199B-08-20 <160> 4 <170> Patentln Ver. <210> <211> <212> <213 1 511

PRT

Human immunodeficiency virus type 1 <400> 1 WO 00/11035 WO 0011035PCTIUS99/1 6725 2 14 Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp Giu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys His Ile Val Trp Ala Ser Arg Giu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu Leu Giu Thr Ser Giu Gly Gly.Arg Gin Ile Leu Gly Gin Leu Gin Pro Ser Leu Gin Thr Gly Ser Giu Giu Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys Vai His Gin Arg Ile Glu Ile Lys Asp Thr Lys Giu Ala Leu Asp Lys Ile Glu Glu Giu Gin Asn Lys Ser Lys 100 105 110 Lys Lys Ala Gin Gin Ala Ala Ala Asp Thr Gly His Ser Ser Gin Vai 120 125 Ser Gin Asn Tyr Pro Ilie Val Gin Asn Ilie Gin Gly Gin Met Val His 130 135 140 Gin Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Vai Glu 14S 145 150 155S6 160 ivn 00n/i1 mn 14 r%_I IU YY/ I oil Glu Lys Ala Phe Ser Pro Giu Val Ile Pro Met Phe Ser Ala Leu Ser 165 170 175 Giu Gly Ala Thr Pro Gin Asp Leu Asn Thr Met Leu Asn Thr Val Gly 180 185 190 Gly His Gin Ala Ala Met Gin.Met Leu Lys Glu Thr Ile Asn Giu Giu 195 200 205 Ala Ala Giu Trp Asp Lys Val Met Pro Val Met Ala Gly Pro Ile Ala 210 215 220 Pro Gly Gin Met Arg 225 Ser Thr Leu Gin Giu 245 Pro Val Gly Giu Ile 260 Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr 230 235 240 Gin Ile Gly Trp Met Thr Asn Asn Pro Pro Ile 250 255 Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 265 270 Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gin Gly 275 280 285 Pro Lys Giu Pro Phe Arg Asp Tyr Val Arg Phe Tyr Lys Thr Leu Arg 290 295 300 WO 00/11035 Pr"T/ IC/i <172 4 14 Ala Glu Gin Ala Ser Gin Glu Val Lys Asn Trp Met Thr Glu Thr Leu 305 310 315 320 Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu 325 330 335 Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gin Gly Val 340 345 350 Gly Gly Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gin 355 360 365 Val Thr Asn Ser Ala Thr Ile Met Met Gin Arg Gly Asn Phe Arg Asn 370 375 380 Gin Arg Lys Ile Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile 385 390 395 400 Ala Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly 405 410 415 Lys Glu Gly His Gin Met Lys Asp Cys Thr Glu Arg Gin Ala Asn Phe 420 425 430 Leu Gly Lys Ile Trp Pro Ser Tyr Lys Gly Arg Pro Gly Asn Phe Leu 435 440 445 Gin Ser Arg Pro Glu Pro Thr Ala Pro Pro Phe Leu Gin Ser Arg Pro 450 455 460 WO) 00/11 035 AfTfI'n1£1f 4U- 1UYIO Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg Ser Gly Val Glu Thr 465 470 475 480 Thr Thr Pro Ser Gin Lys Gin Glu Pro Ile Asp Lys Glu Leu Tyr Pro 485 490 495 Leu Thr Ser Leu Arg Ser Leu.Phe Gly Asn Asp Pro Ser Ser Gin 500 505 510 <210> <211> <212> <213> 2 511

PRT

Orientia tsutsugamushi <400> 2 Ser Ala Met Ser Ala Leu Ser Leu Pro Phe Ser Ala Ser Ala Ile Giu 1 5 10 Leu Gly Glu Glu Gly Leu Glu Cys Gly Pro Tyr Ala Lys Val Gly Val 25 Val Giy Gly Met Ile Thr Gly Val Giu Ser Ala Arg Leu Asp Pro Ala 40 Asp Ala Giu Gly Lys Lys His Leu Ser Leu Thr Asn Gly Leu Pro Phe 55 WO n001103 n PC/I tCO/1 7 2 6 14 Gly Gly Thr Leu Ala Ala Gly Met Thr Ile Ala Pro Gly Glu Arg Ala Glu Ile Gly Val Met Tyr Leu Thr Asn Ile Thr Ala Gin Val Glu Glu Gly Lys Val Lys Ala Asp Ser Val Gly Glu Thr Lys Ala Asp Ser Val 100 105 110 Gly Gly Lys Asp Ala Pro Ile Arg Lys Arg Phe Lys Leu Thr Pro Pro 115 120 125 Gin Pro Thr Ile Met Pro Ile Ser Ile Ala Val Arg Asp Phe Gly Ile 130 135 140 Asp Ile Pro Asn Gin Thr Ser Ala Ala Ser Thr Ser Arg Ser Leu Arg 145 150 155 160 Leu Asn Asp Glu Gin Arg Ala Ala Ala Arg Ile Ala Trp Leu Lys Asn 165 170 175 Cys Ala Gly Ile Asp Tyr Arg Val Lys Asn Pro Asn Asp Pro Asn Gly 180 185 190 Pro Met Val Ile Asn Pro Ile Leu Leu Asn Ile Pro Gin Gly Asn Pro 195 200 205 Asn Pro Val Gly Asn Pro Pro Gin Arg Ala Asn Pro Pro Ala Gly Phe 210 215 220 wn 00n/ 1103 iPT/IC9/1 6725e 7 14 Ala Ile His Asn His Glu Gin Trp Arg His Leu Val Val Gly Leu Ala 225 230 235 240 Ala Leu Ser Asn Ala Asn Lys Pro Ser Ala Ser Pro Val Lys Val Leu 245 250 255 Ser Asp Lys Ile Thr Gin Ile.Tyr Ser Asp Ile Lys His Leu Ala Asp 260 265 270 Ile Ala Gly Ile Asp Val Pro Asp Thr Ser Leu Pro Asn Ser Ala Ser 275 280 285 Val Glu Gin Ile Gin Asn Lys Met Gin Glu Leu Asn Asp Leu Leu Glu 290 295 300 Glu Leu Arg Glu Ser Phe Asp Gly Tyr Leu Gly Gly Asn Ala Phe Ala 305 310 315 320 Asn Gin Ile Gin Leu Asn Phe Val Met Pro Gin Gin Ala Gin Gin Gin 325 330 335 Gly Gin Gly Gin Gin Gin Gin Ala Gin Ala Thr Ala Gin Glu Ala Val 340 345 350 Ala Ala Ala Ala Val Arg Leu Leu Asn Gly Asn Asp Gin Ile Ala Gin 355 360 365 Wn A/ I hlfl 8 14 PC 1/uS9WIj6 72 Leu Tyr Lys Asp Leu Val Lys Leu Gin Arg His Ala Gly Ile Lys Lys 370 375 380 Ala Met Giu Lys Leu Ala Ala Gin Gin Glu Giu Asp Ala Lys Asn Gin 385 390 395 400 Gly Glu Gly Asp Cys Lys Gin Gin Gin Gly Thr Ser Glu Lys Ser Lys 405 410 415 Lys Giy Lys Asp Lys Giu Ala Giu Phe Asp Leu Ser Met Ile Val Gly 420 425 430 Gin Val Lys Leu Tyr Ala Asp Val Met Ile Thr Glu Ser Val Ser Ile 435 440 445 .Tyr Ala Gly Val Gly Ala Gly Leu Ala Tyr Thr Ser Gly Lys Ile Asp 450 455 460 Asn Lys Asp Ile Lys Gly His Thr Gly Met Vai Ala Ser Gly Ala Leu 465 470 475 480 Gly Vai Ala Ile Asn Ala Ala Glu Gly Val Tyr Val Asp Ile Glu Giy 485 490 495 Ser Tyr Met Tyr Ser Phe Ser Lys Ile Giu Giu Lys Tyr Ser Ile 500 505 510 <210> 3 WO 00/11035 9 14 WO 0011035PCT/US99/1 6725 '211> 417 '<212> PRT '213> Human immunodeficiency virus type 1 .<400> 3 Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Giu Leu Asp Arg Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys His Ilie Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu Leu Giu Thr Ser Glu Gly Gly Arg Gin Ile Leu Gly Gln Leu Gln Pro Ser Leu Gin Thr Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys Val His Gin Arg Ile Glu Ile Lys Asp Thr Lys Giu Ala Leu Asp Lys Ile Giu Glu Giu Gin Asn Lys Ser Lys 100 105 110 Lys Lys Ala Gin Gin Ala Ala Ala Asp Thr Giy His Ser Sex- Gin Vai 115 120 125 WO) 00/11035 1DCTfI1CflQ/1A'7')C 10 14 .t3fltO Ser Gin Asn Tyr Pro Ile Val Gin Asn Ie Gin Gly Gin Met Val His 130 135 140 Gin Aia Ilie Ser Pro Arg Thr Leu Asn Ala Trp, Val Lys Vai Val 145 150 155 Giu 160 Giu Lys Aia Phe Ser Pro Giu Val Ile 165 Glu Gly Ala Thr Pro Gin Asp Leu Asn 180 185 Pro Met Giu Ser Ala Leu 170 175 Ser Thr Met Leu Asn Thr Val Giy 190 Gly His Gin Ala Aia Met Gin Met Leu Lys Giu Thr Ile Asn Giu Giu 195 200 205 Ala Ala 210 Glu Trp Asp Lys Val Met Pro Val Met Ala Gly Pro Ile Ala 215 220 Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser 235 240 Pro Gly Met Arg Giu Pro 225 230 Thr Leu Gin Glu Val Gly Glu Ile 260 Gin Ile Gly Trp Met Thr Asn Asn Pro Pro Ile Pro 245 250 255 Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile 265 270 Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gin Gly Pro 275 280 285 WO 00/l11035 vdCIT ?eon/I <P7,wc I1 14 ,5 J2Zfl Lys Giu Pro Phe Arg Asp Tyr Val Arg Phe Tyr Lys Thr Leu Arg Ala 290 295 300 Giu Gin Ala Ser Gin Glu Val Lys Asn Trp Met Thr Giu Thr Leu Leu 305 310 315 320 Val Gin Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly 325 330 335 Pro Ala Ala Thr Leu Giu Glu Met Met Thr Ala Cys Gin Gly Val Gly 340 345 350 Gly Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gin Val 355 360 365 Thr Asn Ser Ala Thr Ile Met Met Gin Arg Gly Asn Phe Arg Asn Gin 370 375 380 Arg Lys Ilie Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile Ala 385 390 395 400 Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys 405 410 415 Glu WO f~i 00113 d- c'll1£ 12 14 I3 IIL; <210> 4 <211> 418 <212> PRT <213> Orientia tsutsugamushi <400> 4 Ala Asp Ser Val Gly Gly Lys Asp Ala Pro Ile Arg Lys Arg Phe Lys 1. 5 10 is Leu Thr Pro Pro Gin Pro Thr Ile Met Pro Ile Ser Ile Ala Val Arg 25 Asp Phe Gly Ile Asp Ile Pro Asn Gin Thr Ser Ala Ala Ser Thr Ser 40 Arg Ser Leu Arg Leu Asn Asp Glu Gin Arg Ala Ala Ala Arg Ile Ala 55 Trp Leu Lys Asn Cys Ala Gly Ile Asp Tyr Arg Val Lys Asn Pro Asn 70 75 Asp Pro Asn Giy Pro Met Val Ile Asn Pro Ile Leu Leu Asn Ile Pro 90 Gin Gly Asn Pro Asn Pro Val Gly Asn Pro Pro Gin Arg Ala Asn Pro 100 105 110 Pro Ala Gly Phe Ala Ile His Asn His Glu Gin Trp Arg His Leu Val 115 120 125 WO 00/11035 DCTII" loanl /IK71 13 14 .IU7/U4) Val Gly Leu Ala Ala Leu Ser Asn Ala Asn Lys Pro Ser Ala Ser Pro 130 135 140 Val Lys Val Leu Ser Asp Lys Ile Thr Gin Ile Tyr Ser Asp Ile Lys 145 150 155 160 Hlis Leu Ala Asp Ile Ala Gly Ile Asp Val Pro Asp Thr Ser Leu Pro 165 170 175 Asn Ser Ala Ser Val Glu Gin Ile Gin Asn Lys Met Gin Glu Leu Asn 180 185 190 Asp Leu Leu Glu Giu Leu Arg Glu Ser Phe Asp Giy Tyr Leu Gly Gly 195 200 205 Asn Ala Phe Ala Asn Gin Ile Gin Leu Asn Phe Val Met Pro Gin Gin 210 215 220 Ala Gin Gin Gin Gly Gin Gly Gin Gin Gin Gin Ala Gin Ala Thr Ala 225 230 235 240 Gin Giu Ala Val Ala Ala Ala Ala Val Arg Leu Leu Asn Gly Asn Asp 245 250 255 Gin Ile Ala Gin Leu Tyr Lys Asp Leu Val Lys Leu Gin Arg His Ala 260 265 270 Gly Ile Lys Lys Ala Met Glu Lys Leu Ala Ala Gin Gin Giu Giu Asp WO 00/11035 14 14 WO 0011035PCTIUS99/1 6725 275 280 285 Ala Lys Asn Gin Gly Giu Giy Asp Cys Lys Gin Gin Gin Gly Thr Ser 290 295 300 Giu Lys Ser Lys Lys Gly Lys Asp Lys Giu Ala Giu Phe Asp Leu Ser 305 310 315 320 Met Ile Val Giy Gin Val Lys Leu Tyr Ala Asp Val Met Ile Thr Giu 32S 330 335 Ser Vai Ser Ile Tyr Ala Gly Val Gly Ala Giy Leu Ala Tyr Thr Ser 340 345 350 Gly Lys Ile Asp Asn Lys Asp Ile Lys Gly His Thr Gly Met Vai Ala 355 360 365 Ser Gly Ala Leu Gly Val Ala Ile Asn Ala Ala Glu Gly Val Tyr Val 370 375 380 Asp Ile Giu Gly Ser Tyr Met Tyr Ser Phe Ser Lys Ile Glu Glu Lys 385 390 395 400 Tyr Ser Ile Asn Pro Leu Met Ala Ser Val Ser Val Arg Tyr Asn Phe 405 410 415 Gin Thr

Claims (11)

1. Isolated scrub typhus antibodies which cross-react with HIV-1 proteins.

2. Antibodies of claim 1 which neutralize HIV-1 virus.

3. A vaccine for passive immunisation against HIV infection comprising an antibody of claim 1.

4. A method for prevention or treatment of HIV infection comprising administration of the vaccine of claim 3. A vaccine for passive immunization against HIV infection comprising an antibody of claim 2.

6. A method for prevention or treatment of HIV infection comprising administration of the vaccine of claim

7. An immunogenic composition for active immunization against HIV infection comprising a peptide or peptides 0. tsutsugamushi which induce an immunogenic Sresponse directed against HIV-1. 15 8. The peptide of claim 7 which is set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or a O *o fragment thereof.

9. A method for prevention or treatment of HIV infection comprising administration of the isolated scrub typhus antibodies of claim 1.

10. A method for prevention or treatment of HIV infection comprising administration 20 of the antibodies of claim 2.

11. The isolated scrub typhus antibodies of claim 1 that recognize HIV-1 p24 antigen S. and/or HIV-1 p17 antigen. S

12. The isolated scrub typhus antibodies of claim 11 that recognize HIV-1 p 17 antigen. P:\OPER\Pk\2388474-183claims.doc-22/08/03 -26-

13. A vaccine for passive immunization against HIV infection comprising an antibody of claim 11. DATED this 22nd day of August 2003 George H. WATT by Davies Collison Cave Patent Attorneys for the Applicant o