WO2012139099A2 - Herpes simplex virus vaccine - Google Patents

Abstract

The present invention relates, in general to herpes simplex virus (HSV) and, particular, to antibodies that are specific for glycoprotein D (gD) of HSV. The invention also relates to prophylactic and therapeutic uses of such antibodies.

Description

HERPES SIMPLEX VIRUS VACCINE

This application claims priority from U.S. Provisional Application

No. 61/473,666; filed April 8, 2011, the entire content of which is incorporated herein by reference.

This invention was made with government support under Grant No. CHAVI U19 AI067854 awarded by the National Institutes of Health. The invention was also made with support from the U.S. Military HIV Research Program (MHRP), Department of Defense, The government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to herpes simplex virus (HSV) and, in particular, to a vaccine against HSV and to a method of inducing an immune response against HSV in a subject using same.

BACKGROUND

HSV types 1 and 2 are enveloped DNA viruses of the herpesvirus family that are common causes of human disease. HSV-1 is frequently acquired early in life such that -50% of 5-year-old children in the US have evidence of infection. Acquisition continues throughout life and 70-90% of the elderly have evidence of prior infection. HSV-2 acquisition is more sporadic with infection rates increasing throughout adolescence and data shows that -20% of US adults have evidence of infection, although, in certain populations, the rates can be substantially higher, in some cases up to 80%.

Herpesvirus infections are acquired through person-to-person contact and the site of entry is skin and/or mucous membranes. The viruses bind to cellular receptors via proteins expressed on the surface of virions, including gD, and interaction of these virus receptors with host receptors triggers the events of virus fusion and host cell infection. Once infection is established in the host, the virus can infect multiple cell types and can cause disease ranging from localized blistering (vesicles), such as is seen in a cold sore, local spread of vesicular rash, dissemination of the vesicular rash, invasion of the bloodstream, infection of internal organs (including the liver), and infection of the central nervous system (including the brain). More extensive disease is associated with increasing degrees of morbidity and mortality.

Once infection has occurred, all herpesvirus infections establish latency in the host. HSV-1 and HSV-2 infect nerve cells, typically peripheral ganglia, and can remain dormant for days to years. Reactivation occurs following signaling events that are poorly understood. Once reactivation occurs, the virus replicates and either asymptomatic shedding of the virus or shedding in the context of disease manifestations can occur. It is these periods of virus replication that are associated with the common manifestations of recurrent HSV disease, including cold sores around the mouth and outbreaks of genital herpes. During periods of such outbreaks, transmissible virus is shed and while symptomatic outbreaks are associated with higher levels of virus shedding, asymptomatic shedding is known to occur frequently. Studies of adult women infected with genital HSV-2 suggest that there is a 1 in 100 chance on any day of asymptomatic shedding of infectious virus.

While many infections with herpes viruses are asymptomatic in healthy hosts or only cause relatively mild or localized disease, infection in hosts with compromised immune systems can be devastating. In particular, populations at very high risk for disseminated or central nervous system disease include newborn infants, patients with inborn errors of the immune system, patients with acquired immune deficiencies (e.g., HIV infection), patients undergoing chemotherapy for malignancies, and the elderly. Such patients are at risk of more severe primary disease, more severe recurrent disease, difficulty controlling infection once established, shorter periods of latency compared to healthy hosts, increased rates of asymptomatic shedding, and a higher likelihood of dissemination.

The immune response to HSV involves innate and adaptive immunity. As with all viral infections, both cell-mediated and humoral responses are critical. The critical importance of humoral immunity has been suggested by studies of HSV transmission around the time of birth (i.e., perinatal or congenital HSV) where infants bom to women experiencing primary HSV disease are more likely to acquire HSV than infants born to women with recurrent HSV. This is thought to be due to transplacental transfer to the infant of IgG antibodies produced by the mother that provide a degree of protection. For this reason, an effective vaccine that can induce such antibodies and/or human mAbs that can be passively administered could provide protection to infants against this disease.

To date, efforts at producing an effective vaccine against HSV have proven disappointing and no approved, commercially available vaccine exists. The present invention provides a novel approach to inducing in a subject (e.g., a human) an effective immune response against HSV.

SUMMARY OF THE INVENTION

In general, the invention relates to HSV. More specifically, the invention relates to a vaccine against HSV and to a method of inducing an immune response against HSV in a subject (e.g., a human) using same.

Objects and advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Binding antibodies in RV144 were examined in 100 vaccine participants. The level of antibodies to the gD peptide were measured. 100% of the vaccinees had IgG antibodies to the gD peptide that were elicited by vaccination.

Figure 2. IgA binding antibodies in RV144 were examined in 100 vaccine participants. The level of antibodies to the gD peptide were measured. About sixty three percent of the vaccinees had IgA antibodies to the gD peptide that were elicited by vaccination.

Figure 3. The gD peptide contains important sequences for HSV entry. Figure 4. Additional studies relating to gD reactive antibodies. Figure 5. Comparison of the vaccine elicited IgG responses to the gD peptide in RV144 compared to another vaccine (Chiron) that used the whole gD protein

(collaboration with L. Corey, U Washington) and a comparison with naturally infected HSV seropositive individuals, either HSV1 or HSV2 or both HSV1/HSV2. RV144 elicited significantly higher antibody responses to the gD peptide than another vaccine (CHIRON) and also compared to natural HSV infection, indicating that these are unique antibody responses to a functional component needed for HSV entry.

Figure 6. Comparison of the vaccine elicited IgA responses to the gD peptide in RV144 compared to another vaccine (Chiron) that used the whole gD protein

(collaboration with L. Corey, U Washington) and a comparison with naturally infected HSV seropositive individuals, either HSV1 or HSV2 or both HSV1/HSV2. RV144 elicited significantly higher antibody responses to the gD peptide than natural HSV infection, indicating that these are unique antibody responses to a functional component needed for HSV entry.

Figures 7A-7C. gD mAb epitopes mapping from RV 144 subject. (Fig. 7A) Monoclonal antibody Ab5157. (Fig. 7B). Monoclonal antibody Ab5190. (Fig. 7C) Monoclonal antibody Ab5188.

Figure 8. Exemplary immunogens.

Figure 9. Design of vaccine immunogens for induction of neutralizing antibodies against HSV in humans. Constructs can be designed to express an HSV gD epitope N- terminal to, for example: 1) a strong or immunodomint antigen, such as tetanus toxin (TT) fragment C (Fairweather, et al., J. Bacteriol. 165:21-27,1986), 2) a weak antigen, such as Human T-cell lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein (Schulz et al, Virology, 184:483-491 , 1991), 3) a non-immunogenic protein, such as human serum albumin (HSA) (Carter, D.C. and He, X.M. Science 249:302-303,1990), or 4) fragments of these proteins. In addition, a linker sequence such as LLE or other amino acids in any order and lengths can be present between HSV gD and these protein sequences.

DETAILED DESCRIPTION OF THE INVENTION

The efficacy seen in the RV144 ALVAC prime gpl20 B/E boost clinical trial demonstrated that a protective vaccine could be made (Rerks-Ngarm, S et al NEJM 361 : 2209-30 (2009)). However, the efficacy was modest at 31 % and the duration of protection short, demonstrating the need for improvement in level of protection (Rerks- Ngarm, S et al NEJM 361 : 2209-30 (2009)). To improve on the efficacy of RV144 results, it was critical to understand the nature of the immunogens in RV144 and to understand why the trial worked, and to define any immune correlates of protection in the trial.

An HSV gD epitope included in the protein boost immunogen of RV144 resulted in enhanced exposure of multiple antibody binding sites that improved the quality of the protein for binding to known monoclonal antibodies (VRCOl , CHOI , A32 mAbs) in comparison with the envelope protein without the gD epitope. Moreover, this immunogen induced antibody responses (IgG, (IgG3), and IgA) in the vaccinees that were of higher magnitude to envelope proteins containing the gD epitope. It was found that these enhanced vaccine-elicited responses were the result of conformational changes to the gpl20 induced by the addition of the gD epitope.

Importantly, and as indicated in the Example that follows, it was found that the gD epitope tag itself was immunogenic in the RV144 trial vaccines. One hundred of vaccinees responded to the gD tag with high levels of IgG anti-gD antibody (Fig. 1).

About sixty three percent of the vaccinees had IgA antibodies to the gD peptide that were elicited by vaccination (Fig.2). As shown in Fig. 3, the gD peptide includes sequences important for HSV entry. (See also Figs. 5 and 6.) The present approach to inducing an immune response against HSV in a subject (e.g., a human) results, at least in part, from these findings. In one embodiment, the present invention relates to an immunogen, gpl20 or gpl40 with an N-terminal gpl20 gD (or portion thereof) tag, with or without a LE sequence and with or without the original 12 amino acids of the N-terminus of g l20 (see exemplary immunogens shown in Figure 8 and in Figure 2 of U.S. Prov. Application No. 61/407,299, filed October 27, 1010) The leader sequence can be the HSV leader sequence, the HIV leader sequence or a transmitted founder virus leader sequence with a position 12 histidine. Advantageously, the Env g l20 or g l40 for connecting to gD tag is a transmitted founder virus Env such as 1086.C, 089.C, 63521. B, 6240.B, 040.B or AI C recombinant transmitted founder Env 707-01-069-2 (see sequences in Table 1, in U.S. Provisional Application No. 61/344,622 and in WO 201 1/106100). In addition the 0219.A signature Env of broad Nabs can also be advantageously used. In addition, the gD HSV tag sequence, or portion thereof, can be added N-terminal to consensus sequences such as the group M consensus CON-S gpl40 or gpl20 sequence (Liao et al, Virology 353(2):268 (2006), PCT/US04/30397, U.S. Application Nos. 10/572,638 and 1 1/896,934) or added N-terminal as described to either the gpl40 or the gpl20 of the mosaic Env sequences (PCT/US2009/004664, U.S. Application Nos. 1 1/990,222 and 12/192,015). Finally, the gD sequence can be added to a subunit of the gpl 20, gpl40 or gpl60 Env sequence.

For induction of neutralizing antibody responses in human, constructs can also be designed to express an HSV gD epitope N-terminal to, for example: 1) a strong or immunodomint antigen, such as tetanus toxin (TT) fragment C (Fairweather, et al., J. Bacteriol. 165:21-27, 1986), 2) a weak antigen, such as Human T-cell lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein (Schulz et al, Virology, 184:483-491 , 1991), 3) a non-immunogenic protein, such as human serum albumin (HSA) (Carter, D.C. and He, X.M. Science 249:302-303,1990), or 4) fragments of these proteins. In addition, a linker sequence, such as LLE or other amino acids in any order and lengths, can be present between the HSV gD epitope and such protein sequences. Examples of such constructs are shown in Fig. 9.

As pointed out above, the gD tag (or epitope) can be 28 amino acids in length or a subunit thereof can be used, e.g., a subunit of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 amino acids in length. The subunit can comprise, for example, LPVLDQ.

The gD+ and gpD+ gpl40 envelopes can be formulated as DNAs (Santra S. et al. Nature Med. 16: 324-8, 2010), for example, rAdenovirus vectored Envs (Barouch DH, et al. Nature Med. 16: 319-23, 2010), recombinant mycobacteria (ie BCG or M smegmatis) (Yu, JS et al. Clinical Vaccine Immunol. 14: 886-093, 2007; ibid 13: 1204-11 , 2006), recombinant vaccinia type of vectors (Santra S. Nature Med. 16: 324-8, 2010). The gD+ envelopes can also be administered as a protein boost in combination with a variety of vectored Env primes (Barefoot B et al. Vaccine 26: 6108-18, 2008), or as protein alone (Liao HC et al Virology 353 : 268-82, 2006). The protein can be administered advantageously with existing adjuvants such as MF59, AS01B or alum and administered either subcutaneously or intramuscularly. Alternatively, the protein or vectored Env can be administered mucosally such as intranasal immunization or by other mucosal routes (Torrieri DL et al Mol. Ther. Oct. 19 2010, E put ahead of print).

Immunogens of the invention are suitable for use in generating an immune response in a patient (e.g., a human patient) to HSV. The mode of administration can vary with the immunogen, the patient and the effect sought, similarly, the dose administered. As noted above, typically, the administration route will be intramuscular, subcutaneous injection (intravenous and intraperitoneal can also be used). Additionally, the formulations can be administered via the intranasal route, or intrarectally or vaginally as a suppository-like vehicle. Optimum dosing regimens can be readily determined by one skilled in the art. The immunogens are preferred for use prophylactically, however, their administration to infected individuals may reduce viral load.

The present invention relates to novel immunogens and compositions and to novel methods of inducing an immune response. Thus, in certain embodiments, the invention includes the immunogens described above, with the proviso that immunogens previously described and/or utilized in connection with the Thai RV144 trial are not included.

Certain aspects of the invention are described in greater detail in the non-limiting Example that follows. (See also PCT/US07/07399, filed March 26, 2007, U.S.

Application No. 12/225,541 , filed September 24, 2008, PCT/US2010/002770, filed

October 18, 2010, U.S. Provisional Application No. 61/407,299, filed October 27, 2010 and Rerks-Ngarm et al, NEJM 361 :2209-30 (2009)). See also U.S. Provisional

Application No. 61/473,543, filed April 8, 201 1 , the entire content of which is incorporated herein by reference.) EXAMPLE

A heterologous prime-boost strategy (canarypox prime, Env protein boost) demonstrated positive results in an efficacy trial (RV144) (Rerks-Ngarm, S et al NEJM 361 : 2209-30 (2009)). Although the immune correlates of protection for the heterologous prime-boost RV144 efficacy trial are as yet undefined, the reduced rate of acquisition without a significant effect on initial viral loads or CD4⁺ T cell counts, have raised the hypothesis of an RV144 vaccine-elicited transient protective B cell response (Rerks- Ngarm, S et al NEJM 361 : 2209-30 (2009)). The duration of protection was short, demonstrating the need for improvement in the level of protection. Thus, to advance vaccine design beyond the RV144 results, it is critical to understand the nature of the immunogens in RV144 in terms of their antigenicity and immunogenicity.

To begin to define correlates of protection, an analysis was made of the the sequence of the envelope immunogens used in the Thai RV144 trial. It was found that the HIV-1 gpl20 Env proteins in the B/E boosts had a herpes simplex virus (HSV) gD protein 28 aa epitope tag and an extra LE aa sequence just after the tag at the N-terminus of g l20. This extra region put on the HIV Env as a tag for purification of the HIV-1 envelope protein contains the HSV gD binding sites for three host receptors for HSV (Connolly et al, J. Virol. 79: 1282 (2005), Yoon et al, J. Virol. 77:9221 (2003), WuDunn and Spear, J. Virol. 63:52 (1989), Connolly et al, J. Virol. 77:8127 (2003), Campadelli- Plume et al, Rev. Med. Virol. 17:313 (2007)).

Due to the positioning of the gD epitope at the base of the g l20 Env, the effect of the gD epitope on the conformations of the neutralizing antibody epitopes within gpl20 was measured. The quaternary V2,V3 antibodies CHOI , CH02, CH03, CH04, and CH05 mAbs (Bonsignori and Haynes, AIDS Research Human Retroviruses P04.52LB (2010)) could bind to the A244, gD+ gpl20 Thai trial immunogen , implying that the A244 gD+ Env was in a similar conformation to gpl20 in the native Env trimer on a virion (Walker et al, Science 326:285 (2009)). The reverted unmutated ancestor antibodies (RUAs) of the CHOI , CH02 and CH03 anti-gpl20 quarternary human mAbs also bound to gD+, A244 gpl 20 Env suggesting that the Thai trial immunogen could bind to the germline B cell receptors of na^'ive B cells.

To determine how inclusion of the gD epitope in the gpl20 Env immunogen impacted the conformation of Env gpl20, a panel of human and mouse monoclonal antibodies (Gao et al, Virology 394:91 (2009)) was used to probe the antigenicity of the Env. In addition to using the A244,gD+, gpl20 envelope protein used in the Thai trial, A244,gD+, gpl20 and A244, gD- gpl20 were constructed and these proteins were expressed in 293T cells (Liao etr al, J. Virol. Methods 158: 171 (2009)). Where the gD tag is in the gpl40 unliganded trimer has been modeled. As expected a monoclonal antibody (Mab 13D7) that binds to the initial 14 N-terminal aa of gpl20 binds only to those gpl20 Env proteins that do not contain the gD epitope and it does not bind to either the A244 gD+ gpl20 produced in CHO cells and used in the Thai trial, nor to the A244 gD+ gpl20 produced in 293T cells. However, the T8 mAb that binds to the CI region of gpl20 is in the gD+ versions of gpl20 and that mAb binding is enhanced to the gD+ gpl20 A244 Envs. gpl20 binding to sCD4 was also markedly enhanced in gpl20s expressing the gD epitope. To confirm this finding, a determination was made of the ability of either A244 gD+ or gD- gpl 20 Env to compete for the anti-CD4 mab Leu 3a that binds to the site on CD4 to which gpl20 binds. It was found that the gD+ but not the gD- A244 293T gpl20 could compete with the anti-CD4 mAb for binding to CD4 on the surface of T cells indicating that the gD tag conferred on A244 the ability to bind native CD4. Next, to determine if the neutralizing epitope of the CD4 binding site was exposed on the surface of gpl20 by the presence of the gD tag, VRCOl mAb binding was tested. The VRCOl mAb, the most potent neutralizing antibody known for HIV- 1 , binds to the CD4 binding site region on the HIV-1 Env in a manner that is most similar to the CD4 molecule (Wu et al, Science 329:856 (2010), Zhou et al, Science 329:81 1 (2010)).

Consistent with enhanced CD4 binding site exposure of the Thai trial immunogen, VRCOl mAb had increased binding to A244 gpl 20 Env that contained the gD epitope compared to the Env without the gD epitope. The dissociation constant ( d) of binding of VRCOl mAb for the A244 gD+ gpl20 was 41 nM compared to a Kd of 164nM for the same envelope without the gD epitope. In contrast, the binding of the anti-V3

monoclonal antibody 19b to both A244 gD+ and A244 gD- gpl20s was identical.

Another potent epitope on gpl20 for broad neutralizing antibodies is the quaternary epitope involving the V2 and V3 regions of g l20 (Walker et al, Science 326:285 (2009)). Both the CHOI -05 (Bonsignori and Haynes, AIDS Research Human Retroviruses P04.52LB (2010)) and the PG9 and PG16 antibodies (Walker et al, Science 326:285 (2009)) bind to this epitope but likely in slightly different orientations. The question asked was whether the presence of the gD tag on A244 Env had any effect on the ability of either CHOI or PG9 to bind to A244 gpl20. It was found that CHOI binding was markedly enhanced to gD+ A244 vs. gD- A244 gpl20. Similarly, when a study was made of PG9 binding to gD+ vs. gD-gpl20 A244 by surface plasmon resonance, it was found that there was marked enhancement in the binding affinity of PG9 to A244 gpl20 by the presence of the gD tag by almost an order of magnitude.

The next question was whether the IgG antibody responses elicited by vaccination with gD+ A244 gpl20 bind better to those gpl 20s that have the gD tag vs those gpl20s that do not. It was found that in both the RV135 (Karnasuta et al, Vaccine 23:2522 (2005)) and RV144 clinical trial vaccinee samples (Rerks-Ngarm, S et al NEJM 361 : 2209-30 (2009)) anti-Env specific IgG antibodies bound better with higher EC50 titers to those gpl20s with gD (i.e., gD+ MN gpl20, gD+ A244 gpl20, gD+ 92TH023 gpl20, and gD+ GNE8 gpl20) compared to those gpl20 Envs without gD tag (i.e. gD- MN gpl20, gD- A244 gpl20, and gD- 92TH023 gpl20). Of note, IgG3 antibodies had a pronounced difference in the concentration of antibodies circulating in the plasma of vaccinees that preferentially bound to gD+ gpl20 Envs.

It was next asked if the gD epitope tag itself was immunogenic in the RV144 trial vaccinees and it was found that 100% of vaccinees responded to the gD tag with high levels of IgG anti-gD antibody (Fig. 1). To further probe the epitope specificity of the vaccine-elicited IgG responses, antibody blocking assays were used. Approximately 30%> of vaccinees had antibody responses that blocked A32 mAb. The gD+ A244 gpl20s had markedly enhanced expression of the A32 epitope compared to the gD-, A244 gpl20. Antibodies that could block lb 12 mAb were detected and the blocking was higher in Env proteins containing the gD epitope. There were no significant antibody responses that blocked 2G12 mAb nor 27G2 mAb binding. The linear epitopes recognized by vaccine elicited IgG responses were against the V2, V3, CI and C5 regions of gpl20 Env epitopes of multiple clades.

Although there were no mucosal specimens available to examine the mucosal response to vaccination, a measurement was made of IgA responses to the vaccine immunogens. Eighty-nine percent of vaccinees had IgA responses to the vaccine strain MN and twenty-three percent had IgA responses to a primary clade A gpl20 (OOMSA). Significantly higher IgA responses were detected against the MN g l20 containing the gD epitope compared to MN gpl20 without the gD epitope. Measurement was also made of IgA responses elicited directly to the gD epitope, and it was found that a significant proportion, sixty-three percent, of vaccinees had IgA responses to the HSV gD epitope (Fig 2).

To address the quality of the vaccine elicited IgG response, the avidity of the purified plasma IgG to a series of envelopes was measured. It was found that several subjects demonstrated binding antibody breadth to a clade A isolate were also among those subjects that had the highest avidity for the vaccine strain immunogen and had the highest neutralizing antibody titers. A critical question to address was whether the enhanced binding of vaccinee serum to gD+ Env gpl20s was due solely to an additive effect of the presence of anti-gD antibodies. Alternatively, it was hypothesized that the gpl20 gD+ protein was in an optimal conformation to induce gpl 20 antibody

specificities that could not bind (or bound less well) to gD- gpl 20. Subjects with high avidity to A244gD+ with low avidity to gD epitope, and the inability to block the binding the A244 gD+ Env with the gD Fab were studied. In one subject that had the highest neutralizing antibody responses, and the highest binding to a primary Clade A isolate, the A244 gpl20 gD+ binding activity of the purified plasma IgG was blocked by CHOI mAb and A32 mAb. Thus, the RV144 immunogen containing the gD epitope tag elicited antibody responses that were specifically induced by the inclusion of the gD sequence in the gpl20 protein immunogen. The gD epitope tag induced epitopes in the gpl20Env are highly functional sites to which antibodies can bind and mediate ADCC (A32), and neutralization (CHOI , VRCOl). All documents and other information sources cited herein are hereby incorporated entirety by reference.

Table 1

>B.63521 gpl40C

MRWGIRKNYQHLWRWGTMLLGILMICSAAAQLWWYGVPWKEATTTLFCASDAKAYDTEW1W

ATHACVPTDPNPQELVLAl TENFlSniWNNTIWEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDVTNA

TNINATNINNSSGGVESGEIKNCSFNITTSVRDKVQKEYALFYKLDIVPITNESSKYRLISCNTSVLT

QACPKVSFEPIPIHYCAPAGFAILKCNNETFNGKGPCINVSTVQCTHGIRPWSTQLLLNGSLAEKEV

IIRSDNFSDNAKNIIVQLKEYWINCTRPNNNTRKSIHIGPGRAFYATGEIIGNIRQAHCNISRSKWN

DTLKQI AKLGEQFRNK IVFNPSSGGDLEIVTHSFNCGGEFFYCNTTKLFNSTWIREGNNGTWNG I

GLNDTAGNDTIILPCKIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLILTRDGGKDDSNGSEILEI

FRPGGGD RDNWRSELYKYKVVRIEPLGVAPTRAR1?RWQKEK£AVGLGA FLGFLGAAGSAMGAASM

TLTVQARQLLSGIVQQQNNLLRAIEAQQHMLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLI

CTTDVPWDTSWSNKTLDDIWGSNMTWMEWEREIDNYTSTIYTLLEEAQYQQEKNEKELLELDKWASLW

NWFDITNWLWYIR*

>B.6240 140C

MRWGIRKNYQHLWRWGIWRWGIMLLGTLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYSPE

KHNIWATHACVPTDPNPQELVLGNVTEDFNMWKNNWEQ HEDIISLWDQSLKPCVKLTPLCVTLNCT

DLKNSATDTNGTSGTNNRTVEQGMETEIKNCSFNITTGIGNKMQKEYALFYKLDWPIDSNNNSDNTS

YRLISCNTSWTQACPKTSFEPIPIHYCAPAGFAILKCNNKTFSGKGPCKNVSTVQCTHGIRPWSTQ

LLLNGSLAEEEIVIRSENFTNNAKTIIVQLNESVIINCTRPNNNTRKGIHIGLGRALYATGDIIGDIR

QAHCNLSSKSWNKTLQQWRKLREQFGNKTIAFNQSSGGDQEIVKHSFNCGGEFFYCDTTQLFNSTWS

SNDT NSTGVQDNNITLPCRIKQIINiWQEVGKAMYAPPIQGLISCSSNITGLLLTRDGGTNNTNATE

IFRPGGGD RDNWRSELYKYKWKIEPLGIAPTKAK.ERWQREKFAVGLGAVFIGFLGAAGSTMGAAS

VTLTVQARQLLSGIVQQQNNLLRAIEAQQHMLQLTVWGIKQLQARILAVERYLKDQQILGIWGCSGKL

ICPTAVPWNASWSNKSLTAIWNNMTWMEWEREIDNYTGLIYSLIEESQIQQEQNEKELLELDKWASLW

NWFDITKWLWYIK*

>C.1086C_140C

MRVRGIWKHWPQWLIWSILGFWIGNMEGSWTWYGVPWKEAKTTLFCASDAIiAYEKEVHNVWATHA CVPTDPNPQEMVLANVTENFN AJKND VEQMHEDIISLWDESLKPCVKLTPLCVTLNCTNVKGNESDT

SEVMKNCSFKATTELKDKKHKVHALFYKLDWPLNGNSSSSGEYRLINCNTSAITQACPKVSFDPIPL HYCAPAGFAILKCNNKTFNGTGPCRNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTNNAKT IIVHLNESWIVCTRPNNNTRKSIRIGPGQTFYATGDIIGNIRQAHCNINESKWNNTLQKVGEELAKH FPSKTIKFEPSSGGDLEITTHSFNCRGEFFYCNTSDLFNGTYRNGTYNHTGRSSNGTITLQCKIKQII NMWQEVGRAIYAPPIEGEITCNSNITGLLLLRDGGQSNETNDTETFRPGGGDMRDNWRSELYKYKWE

IKPLGVAPTEAK£RWEREK£AVGIGAVFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQSNLLRA IEAQQHMLQLT GIKQLQARVLAIERYLKDQQLLGMWGCSGKLICTTAVP NSSWSNKSQNEIWGNM TWMQWDREINNYTNTIYRLLEDSQNQQEKNEKDLLALDSWKNLWNWFDISKViLWYIK*

>C089C_140C

MRVRGMLRNCQQFRMIWGILGFWMLMICSWGNLWT YGVP KFAKTTLFCASDARAYEREVH]SN/VI ATHACVPTDPNPQEWLVWTENFNMG^

TNNGSVIVVNENSTMYGEIQNCSFKVNSEIKGKKQDVYALFNSLDIVKLYNNGTSQYRLINCNTSTLT QACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEGEI IIRSK LTDNTKTIIVHLNESIKINCIRPNNNTRRSIRIGPGQAFYAANGIVGNIRQAHCNISEGEWN KTLYRVSRKLAEHFPGKEIKFKPHSGGDLEITTHSFNCRGEFFYCNTSKLFNGTYNGTYTNNDTNSTI ILPCRIKQIINMWQEVGQAMYAPPIEGIIACNSTITGLLLTRDGGDKNGSKPEIFRPGGGDMRDNWRS ELYKYKWEIKPLGIAPTKAKERWEKEKTIQKEAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLL SGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVLAMERYLQDQQLLGIWGCSGKLICTTAVPWNSS WSNKTLEYIWGNMTMQWDREIDNYTGIIYDLLEDSQIQQEKNEKDLLALDSWKNLWSWFSITMAJLWY

IK*

>A1CD.707010629_B5_140C

MKVRGTQRNYQNLWRWGILGFVMLIICSAAEHLWTWYGVPWJKDAKTTLFCASDAKAYDTEVHNVV/ ATHACVPTDPNPQEIILKWTENFNVWKNDMVEQMHQDIISLWDQSLKPCVKLTPLCVTLDCHNITTP PSNNTGNITSNTTIGNNTNGDNKTYDINMEMTNCSFNATTWRDKKQKVYSLFYKLDIVPIDEDNNSS KSNSTQYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCRDTKFNGTGPCKNVSTVQCTHGIR PWSTQLLLNGSLAEEEVIIRSENISDNAKTIIVQFTKPVSINCTRPSNNTRKGVHLGPGQVLYATGG IIGDIRQAHCNVSRKDWEEALKNVTTQLGKHFNTTIIFTSPSGGDLEITTHSVNCAGEFFYCNTSGLF NSTWSTNGNWTQNETDNKTETISLPCRIKQIINiWQRVGQAMYAPPIQGVISCNSSITGLLLTRDGG YNNSNNETFRPVGGNMKDNWRSELYKYKVWIEPLGIAPTRAKRBVVEREKEAVGFGALFLGFLGAAG STMGAASITLTVQARQLLSGIVQQQNNLLRAIEAQQHMLQLTVWGIKQLQARVLAVERYLKDQQLLGI WGCSGKLICTTWPWSJSSWSNKSHDEIWE3 T¾MQWEREIDNYTSTIYWLLEVSQTQQEK EQDLLAL DKWA LWTWFDI WLWYIK*

>A.0219_140C

MRVMGTQRNYPNLWRWGTMLFLGIIICSAAENLWVNVYYGVPVWKEAETTLFCASDAKAYS EAHNVW

ATHACVPTDPNPQEWLENVTEEFNWRNKMVDQMQEDIASLWDQFLQPCVKLTPLCVTLNCSNPKNP

DNSTDNSTGIGREDMKDMKNCSFNMTTELRDKHQKMYSLFYRLDIEELNENSNSSNSSSSNSSEYRLI

NCNTSTIAQACPKVSFEPIPIHYCAPAGFAILKCRDKKFNGTGPCRNVSTVQCTHGIKPWSTQLLLN

GSLAEKGIKIRSENISESAKTIIVQLDQPWINCTRPNNNTRTSIPMGPGRALYATGAITGDPRQAHC

NISREKWNETLSKVAKKLKEYFNNRTIIFTNASGGDVEVTTHSFNCGGEFFYCNTSNLFNSTWGNGSY

STNDTGDANSNITIQCRIKQIVRMWQRTGQAMYAPPIKGIIRCMSNITGLLLTRDGGINRTNETFRPI

GGDMMDNWRSELYKYKWRIEPIGVAPNRAKRiiWEREKEAVFGMGAVFLGFLGAAGSTMGAASITLT

LQARQLLSGIVQQQSNLLRAIEAQQHLLKLTVWGIKQLQARVLAVERYLQDQQVLGLWGCSGKIICAT

NVPWNSSWSNKSYGEIWDNMTWLEWDKEVSNYTDIIYDLIAKSQNQQEKNEQDLLALDKWTSLWGWFE

ISRWLWYIK*

>A.0219_140C_gD+

MGGAAARLGAVILBWIVGLHGVRGKYALAllkSL^

ASDARAYSTEAHNW

TPLCVTLNCSNPKNPDNSTDNSTGIGREDMKDMKNCSFNMTTELRDKHQKMYSLFYRLDIEELNENSN

SSNSSSSNSSEYRLINCNTSTIAQACPKVSFEPIPIHYCAPAGFAILKCRDKKFNGTGPCRNVSTVQC

THGIKPWSTQLLLNGSLAEKGIKIRSENISESAKTIIVQLDQPWINCTRPNNNTRTSIPMGPGRAL

YATGAITGDPRQAHCNISREKWNETLSKVAKKLKEYFNNRTIIFTNASGGDVEVTTHSFNCGGEFFYC

NTSNLFNSTWGNGSYSTNDTGDANSNITIQCRIKQIVRMWQRTGQAMYAPPIKGIIRCMSNITGLLLT

RDGGINRTNETFRPIGGDMMDNWRSELYKYKWRIEPIGVAPNRAKRf EREKfAVFGMGAVFLGFL

GAAGSTMGAASITLTLQARQLLSGIVQQQSNLLRAIEAQQHLLKLTVWGIKQLQARVLAVERYLQDQQ

VLGLWGCSGKIICATNVPWNSSWSNKSYGEIWDNMTWLEWDKEVSNYTDIIYDLIAKSQNQQEKNEQD

LLALDKWTSLWGWFEISRWLWYIK*

>A.0219_120_„gD+

MGGAAARLGAVILFWIVGLHGWGKYAIADASLKi^

ASDAKAYSTEAHNVWATHACVPTDPNPQEWLENVTEEFNMWRN MVDQMQEDIASLWDQFLQPCVKL

TPLCVTLNCSNPKNPDNSTDNSTGIGREDMKDMKNCSFNMTTELRDKHQKMYSLFYRLDIEELNENSN

SSNSSSSNSSEYRLINCNTSTIAQACPKVSFEPIPIHYCAPAGFAILKCRDKKFNGTGPCRNVSTVQC

THGIKPWSTQLLLNGSLAEKGIKIRSENISESAKTIIVQLDQPWINCTRPNNNTRTSIPMGPGRAL

YATGAITGDPRQAHCNISREKWNETLSKVAKKLKEYFNNRTIIFTNASGGDVEVTTHSFNCGGEFFYC

N SNLFNSTWGNGSYSTNDTGDANSNITIQCRIKQIVRMWQRTGQAMYAPPIKGIIRCMSNITGLLLT

RDGGINRTNETFRPIGGDMMDNWRSELYKYKWRIEPIGVAPNRAKRi?WEREK/?*

>A.0219_^140· »12_..12_gD+

MGGAAARLGAVILFWIVGLHGVRGKYALADAS LKM¾DPNRFRGKDl-iP VI.DQLL£AAENLWVNVYYGV

PVWKEAETTLFCASDAKAYSTFAHNVWATHACVPTDPNPQEVYLENVTEEFNMWRNKMVDQMQEDIAS

LWDQFLQPCVKLTPLCVTLNCSNPKNPDNSTDNSTGIGREDMKDMKNCSFNMTTELRDKHQKMYSLFY

RLDIEELNENSNSSNSSSSNSSEYRLINCNTSTIAQACPKVSFEPIPIHYCAPAGFAILKCRDKKFNG

TGPCRNVSTVQCTHGIKPWSTQLLLNGSLAEKGIKIRSENISESAKTIIVQLDQPWINCTRPNNNT

RTSIPMGPGRALYATGAITGDPRQAHCNISREKWNETLSKVAKKLKEYFNNRTIIFTNASGGDVEVTT

HSFNCGGEFFYCNTSNLFNSTWGNGSYSTNDTGDANSNITIQCRIKQIVRMWQRTGQAMYAPPIKGII

RCMSNITGLLLTRDGGINRTNETFRPIGGDMMDNiAjRSELYKYKWRIEPIGVAPNRAKREWEREKEA

VFGMGAVFLGFLGAAGSTMGAASITLTLQARQLLSGIVQQQSNLLRAIEAQQHLLKLTVWGIKQLQAR

VLAVERYLQDQQVLGLWGCSGKIICATNVPWNSSWSNKSYGEIWDNMTWLEWDKEVSNYTDIIYDLIA

KSQNQQEKNEQDLLALDKWTSLWGWFEISRWL YIK*

>C.1086C_140C_gD+

MGGAAARLGAVILFWIVGLHGVRGKYALADASl-JKM^

AS^AKAYEKEVHNWATHACVPTDPNPQEMVLANVTENFNMWKNDMVEQMHEDIISLWDESLKPCVKL

TPLCVTLNCTNVKGNESDTSEVMKNCSFKATTELKDKKHKVHALFYKLDWPLNGNSSSSGEYRLINC

NTSAITQACPKVSFDPIPLHYCAPAGFAILKCNNKTFNGTGPCRNVSTVQCTHGIKPWSTQLLLNGS

LAEEEIIIRSENLTNNAKTIIVHLNESVNIVCTRPNNNTRKSIRIGPGQTFYATGDIIGNIRQAHCNI

NESKWNNTLQKVGEELAKHFPSKTIKFEPSSGGDLEITTHSFNCRGEFFYCNTSDLFNGTYRNGTYNH

TGRSSNGTITLQCKIKQIINMWQEVGRAIYAPPIEGEITCNSNITGLLLLRDGGQSNETNDTETFRPG

GGDMRDNWRSELYKYKWEIKPLGVAPTEAK£RWEREK£AVGIGAVFLGFLGAAGSTMGAASMTLTV

QARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVLAIERYLKDQQLLGMWGCSGKLICTTA

VPWNSSWSNKSQNEIWGNMTWMQWDREINNYTNTIYRLLEDSQNQQEKNEKDLLALDSWKNLWNWFDI

SKWLWYIK*

>C .1086C_120_gD+

TPLCVTLNCTNVKGNESDTSEVMKNCSFKATTELKDKKHKVHALFYKLDWPLNGNSSSSGEYRLINC

NTSAITQACPKVSFDPIPLHYCAPAGFAILKCNNKTFNGTGPCRNVSTVQCTHGIKPWSTQLLLNGS

LAEEEIIIRSENLTNNAKTIIVHLNESWIVCTRP NNTRKSIRIGPGQTFYATGDIIGNIRQAHCNI

NESK N TLQKVGEELAKHFPSKTIKFEPSSGGDLEITTHSFNCRGEFFYCNTSDLFNGTYRNGTY H

TGRSSNGTITLQCKIKQII MWQEVGRAIYAPPIEGEITCNSNITGLLLLRDGGQSNETNDTETFRPG

GGDMRD WRSELYKYKWEIKPLGVAPTEAKRRWEREKR*

>C.1086C gpl40C__«12__gD+

MGGAAARLGAVILF IVGLHGVRGKYJMliA^

PVWKEAK TLFCAS

LWDESLKPCVKLTPLCVTLNCTNVKGNESDTSEVMKNCSFKATTELKDKKHKVHALFYKLDWPLNGN SSSSGEYRLINCNTSAITQACPKVSFDPIPLHYCAPAGFAILKCNNKTFNGTGPCRNVS VQCTHGIK PWSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVNIVCTRPMNNTRKSIRIGPGQTFYATGD IIGNIRQAHCNINESKWNNTLQKVGEELAKHFPSKTIKFEPSSGGDLEITTHSFNCRGEFFYCNTSDL FNGTYR GTYNHTGRSSNGTITLQCKIKQII MWQEVGRAIYAPPIEGEITCNSNITGLLLLRDGGQS NETNDTETFRPGGGD RD WRSELYKYKWEIKPLGVAPTEAK.ERWEREK.EAVGIGAVFLGFLGAAG STMGAAS TLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVLAIERYLKDQQLLGM WGCSGKLICTTAVPWNSSWSNKSQNEIWG MTWMQ DREINNYTNTIYRLLEDSQNQQEK EKDLLAL DSWKNLWNWFDISKWLWYIK*

Note: HIV-1 gpl20-gp41 cleavage site was mutated from the residue Arg (R) to Glu (E) as shown in italics. HSV leader sequence was underlined, HSV gD sequence is bolded and linker sequence between gD and HIV sequences is shown in italics .

Claims

WHAT IS CLAIMED IS:

1. An immunogen comprising a gpl20, gpl40 or gpl 60 HIV- 1 Env sequence, or subunit thereof, and an N-terminal herpes simplex virus (HSV) gD tag sequence, or portion thereof, and, optionally, an LE sequence and, optionally, the 12 amino acids of the N-terminus of gpl20.

2. The immunogen according to claim 1 wherein said immunogen further comprises a leader sequence.

3. The immunogen according to claim 2 wherein said leader sequence is an HSV leader sequence, an HIV leader sequence or a transmitted founder virus leader sequence with a position 12 histidine.

4. The immunogen according to claim 1 wherein the gpl20, gpl40 or gpl60 Env sequence is a transmitted founder virus Env sequence.

5. The immunogen according to claim 4 wherein the Env sequence is 1086.C, 089.C, 63521 ,B, 6240.B, 040.B or AlC recombinant transmitted founder Env 707-01-069-2.

6. The immunogen according to claim 1 wherein said gD HSV tag sequence, or portion thereof, is present N-terminal to a group M consensus CON-S g l40 or gpl20 sequence or a mosaic Env sequence.

7. An immunogen comprising an HSV gD epitope N-terminal to a strong or immunodominant antigen, a weak antigen, a non-immunogenic protein, or fragment thereof.

8. The immunogen according to claim 7 wherein said immunogen comprises a linker sequence between said HSV gD epitope and said strong or immunodominant antigen, weak antigen, non-immunogenic protein, or fragment thereof.

9. The immunogen according to claim 7 wherein said HSV gD epitope is N- terminal to tetanus toxin (TT) fragment C, Human T-cell lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein, human serum albumin (HSA), or fragment thereof.

10. The immunogen according to claim 7 wherein said immunogen comprises an amino acid sequence shown in Fig. 9.

1 1. The immunogen according to claim 1 , or claim 7, wherein said HSV gD tag sequence, or said HSV gD epitope, comprises the sequence LPVLDQ.

12. A nucleic acid comprising a nucleotide sequence encoding said

immunogen according to claim 1 or 7.

13. The nucleic acid according to claim 12 wherein said nucleic acid is present in a vector.

14. The nucleic acid according to claim 13 wherein said vector is a viral vector.

15. The nucleic acid according to claim 13 wherein said vector is a adenoviral vector, a recombinant mycobacterial vector, or a recombinant vaccinia vector.

16. A method of inducing an immune response against HSV comprising administering to a mammal an amount of said immunogen according to claim 1 or 7 sufficient to effect said induction.

17. The method according to claim 16 wherein a nucleic acid comprising a nucleotide sequence encoding said immunogen is administered so that said nucleic acid is expressed and said immunogen is thereby produced.

18. The method according to claim 16 wherein said mammal is a human.

19. A composition comprising said immunogen according to claim 1 or 7 and a carrier.

20. A composition comprising said nucleic acid according to claim 12 and a carrier.