Functional properties and epitope characteristics of T-cells recognizing natural HIV-1 variants

To understand how broad recognition of HIV-1 variants may be achieved we examined T-cell reactivity in newly infected persons as well as vaccine recipients to a broad spectrum of potential T-cell epitope (PTE) variants containing conservative, semi-conservative and non-conservative amino acid substitutions. Among early infected persons T-cells recognized epitope variants with one substitution at a significantly higher frequency versus those with two (P = 0.0098) and three (P = 0.0125) substitutions. Furthermore T-cells recognized variants containing conservative substitutions at a higher frequency versus those containing semi-conservative (P = 0.0029) and non-conservative (P < 0.0001) substitutions. Similar effects were observed on recognition of variants by vaccine-induced T-cells. Moreover even when variants were recognized, the IFN-γ and granzyme B responses as well as T-cell proliferation were of lower magnitude. Finally, we show that epitope distribution is strongly influenced by both processing preferences and amino acid entropy. We conclude that induction of broad immunity is likely to require immunogen sequences that encompass multiple variants. However, cost-effective design of peptide and sequence based vaccine immunogens that provide maximal coverage of circulating sequences may be achieved through emphasis on virus domains likely to be T-cell targets.     

Mucosal antibodies induced by tandem repeat of 2F5 epitope block transcytosis of HIV-1

Induction of mucosal antibodies to prevent HIV infection is an important strategy for the HIV-1 prophylaxis. Here we report an epitope-vaccine based antigen that was able to elicit mucosal antibodies capable of blocking HIV-1 transcytosis. Because the ELDKWA epitope of neutralizing antibody 2F5 plays a crucial role in transcytosis, a series of immunogens that contain tandem copies of ELDKWA were prepared. Mice were immunized with these immunogens intranasally, and received intraperitoneal + intranasal boosters. The immunogens that contained more ELDKWA epitopes elicited higher level of mucosal ELDKWA-epitope specific IgAs and systemic IgGs. Although the antisera from the immunized mice exhibited mild neutralizing potency to HIV-1 isolates HXB2 and JRFL, the affinity purified mucosal ELDKWA-epitope specific antibodies could block the transcytosis of cell-free CNE3 (a primary isolate of subtype CRF01_AE) in human tight epithelial models.     

Env–2dCD4S60C complexes act as super immunogens and elicit potent,broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1)

The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4^S60C are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env–2dCD4^S60C complexes described here are “super” immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4^60C. Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env–2dCD4^S60C complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted.     

A bivalent HCV peptide vaccine elicits pan-genotypic neutralizing antibodies in mice

Vaccine development for antigenically variable pathogens has faltered because extreme genetic diversity precludes induction of broadly neutralizing antibodies (nAB) with classical vaccines. Here, using the most variable epitope of any known human pathogen (HVR1 of HCV), we describe a novel approach capable of eliciting broadly neutralizing antibodies targeting highly variable epitopes. Our proof-of-concept vaccine elicited pan-genotypic nAB against HCV variants differing from the immunogen sequences by more than 70% at the amino acid level. These findings suggest broadly nAB to highly variable pathogens can be elicited by vaccines designed to target physicochemically conserved residues within hypervariable epitopes.     

Expanded breadth of virus neutralization after immunization with a multiclade envelope HIV vaccine candidate

Although the V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) effectively elicits potent neutralizing antibody responses, the specificity of the antibody response is often restricted to T cell line adapted (TCLA) strains and a small subset of primary isolates, limiting its utility for an AIDS vaccine. In this study, we have compared Env immunogens with substituted V3 regions to combinations of strains from different clades and evaluated their ability to expand the breadth of the neutralizing antibody response. When the V3 region from HIV BaL was substituted for HIV HXB2, an effective neutralizing antibody response against several clade B primary isolates was elicited, but it remained restricted to neutralization of most clade B isolates. In an attempt to expand this response further, a linear epitope recognized by the broadly neutralizing 2F5 antibody was inserted into V3. A V3 2F5 epitope was identified that bound to 2F5 and elicited a potent 2F5 antibody response as an immunogen, but the antisera neutralized only a lab-adapted strain and not primary isolates. In contrast, combinations of Envs from clades A, B, and C, elicited neutralizing antibodies to a more diverse group of primary HIV-1 isolates. These studies suggest that combinations of Env immunogens, despite the limited reactivity of the V3 from each component, can be used to expand the breadth of the neutralizing antibody response.     

Antitumor efficacy of MUC1-derived variable epitope library treatments in a mouse model of breast cancer

The identification of novel targets for cancer immunotherapy and the development of new vaccine immunogens are subjects of permanent interest. MUC1 is an overexpressed antigen found in most tumors, and its overexpression correlates with poor prognosis. Many attempts to direct the immune response against MUC1 in tumor cells have failed, including several clinical trials. We have previously developed an innovative Variable Epitope Library (VEL) vaccine platform that carries massively substituted mutant variants of defined epitopes or epitope regions as an alternative to using wild-type peptide sequences-based immunogens. Here, two murine MUC1-derived epitopes equivalent to the previously tested in cancer immunotherapy human MUC1 regions were used to generate VELs. We observed that vaccination with the 23L VEL immunogens, encompassing the entire signal peptide region of MUC1, reduces the tumor area compared to the wild-type sequence treatment. Contrastingly, vaccination with the MUC1 signal peptide-derived predicted CD8⁺+ T cell epitope-based VEL, 9MUC1spL, showed similar tumor area reduction as the wild-type treatment; however, a decrease in lung metastasis after 9MUC1spL treatment was observed. In addition, vaccination induced a large pool of CD8⁺ T cells which recognized most variant epitopes from 9MUC1spL. Also, we generated MUC1 variable number tandem repeat (VNTR)-based VELs that reduced the metastatic burden when dendritic cells and M13 recombinant bacteriophages were used as vaccine carriers. Collectively, our data demonstrate the immunogenic and antitumor properties of MUC1 signal peptide- and VNTR-derived VEL immunogens.     

Role of lipid structure in the humoral immune response in mice to covalent lipid–peptides from the membrane proximal region of HIV-1 gp41

The membrane proximal region (MPR) of HIV-1 gp41 is a desirable target for development of a vaccine that elicits neutralizing antibodies since the patient-derived monoclonal antibodies, 2F5 and 4E10, bind to the MPR and neutralize primary HIV isolates. The 2F5 and 4E10 antibodies cross-react with lipids and structural studies suggest that MPR immunogens may be presented in a membrane environment. We hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomal bilayers. In a comparison of eight lipids conjugated to an extended 2F5 epitope peptide, a sterol, cholesterol hemisuccinate (CHEMS), was found to promote the strongest anti-peptide IgG titers (6.4 × 10⁴) in sera of BALB/C mice. Two lipid anchors, palmitic acid and phosphatidylcholine, failed to elicit a detectable serum anti-peptide IgG response. Association with the liposomal vehicle contributed to the ability of a lipopeptide to elicit anti-peptide antibodies, but no other single factor, such as position of the lipid anchor, peptide helical content, lipopeptide partition coefficient, or presence of phosphate on the anchor clearly determined lipopeptide potency. Conjugation to CHEMS also rendered a 4E10 epitope peptide immunogenic (5.6 × 10² IgG titer in serum). Finally, attachment of CHEMS to a peptide spanning both the 2F5 and 4E10 epitopes elicited serum IgG antibodies that bound to each of the individual epitopes as well as to recombinant gp140. Further research into the mechanism of how structure influences the immune response to the MPR may lead to immunogens that could be useful in prime-boost regimens for focusing the immune response in an HIV vaccine.     

Enhancement of epitope-specific cellular immune responses by immunization with HIV-1 peptides genetically conjugated to the B-subunit of recombinant cholera toxin

As more HIV-1 infected patients receive anti-retroviral drug treatment, the occurrence of drug-resistant variants of the virus is increasing. We have previously shown that mutated HIV peptide sequences represent mutations induced by antiretroviral drugs are equally good and often better immunogens than wild type peptides. The non-toxic B subunit of cholera toxin (CTB) is an active substance in the oral cholera vaccine, and has been shown to bind ganglioside receptors and activate mucosal cells. By fusing mutant epitopes deriving from HIV-1 enzymes with the B subunit of cholera toxin, we aim is to induce cellular responses against virus harboring drug-induced mutations. We successfully created conjugates of HIV peptide sequences fused to rCTB. The immune response against the different peptides was strongly enhanced by the fusion to the toxin. Moreover, immunization with sequence containing drug-induced mutation triggered a cross-reactive immune response against the wild type epitope. Long-term follow-up of immunized animals revealed a persistence of cellular immune response for over 4 months, which could readily be boosted with an additional late immunization. By linking HIV-peptides to the B subunit of cholera toxin it is thus possible to stimulate a strong and long-lasting immune response, significantly better than that evoked by the peptide alone.     

Neutralization epitopes of the HIV-1 primary isolate DH012

To study HIV-1 primary isolate neutralization, we have used DH012 as a model to study the immunogenicity of several DH012 immunogens and determine the potential neutralization epitopes in the virus envelope glycoprotein. Previously, we identified that DH012 infected animals mount potent neutralizing activity against a conformational epitope (CEV) that involves multiple variable regions. In this study, we show that the conformational epitope can be reconstituted with one gp120 recombinant fragment containing sequences from the V1/V2 loop and the bridging sheet of gp120 and a V3 peptide. In contrast to DH012 infection, we previously demonstrated that animals immunized with DH012 gp120 induced potent neutralizing antibodies directed at the V3 domain of gp120. In this study, a second neutralizing activity against the V1/V2 region of gp120 was identified from the same guinea pig sera. In summary, several neutralization epitopes are identified on DH012, including the CEV, V1/V2, V3, 17b, IgG1b12, and 2G12 epitopes. Infectious DH012 virus carrying oligomeric envelope appears to raise primarily neutralizing antibodies that recognize a discontinuous conformationally dependent epitope whereas the monomeric gp120 induces antibodies that are primarily directed at epitopes in the V3 and V1/V2 domains. The DH012 neutralizing epitopes, such as V1/V2 and V3, are either cryptic or poorly immunogenic in chimpanzees. However, immunogens, such as gp120, could be designed to induce neutralizing activity against epitopes that are poorly immunogenic, such as V1/V2 of DH012, in the native envelope glycoproteins.     

Quantitative assessment of masking of neutralization epitopes in HIV-1

Despite the frequent observation of masking of HIV-1 neutralization epitopes, its extent has not been previously systematically assessed either for multiple epitopes presented by individual viruses or for individual epitopes across multiple viral strains. Using a recently developed method to identify amino acid sequence motifs required for recognition by HIV-1-neutralizing monoclonal antibodies (mAbs), we visualized the patterns of masking of specific epitopes targeted by mAbs in a diverse panel of HIV-1 isolates. We also calculated a specific masking intensity score for each virus based on the observed neutralization activity of mAbs against the epitopes in the virus. Finally, we combined these data with estimates of the conservation of each mAb-targeted epitope in circulating HIV-1 strains to estimate the effective neutralization potential (E_N) for each mAb. Focusing on the V3 loop of gp120 as a prototype neutralization domain, we found that the V3 loop epitope targeted by mAb 2219 is one of the least masked mAbs and it has the highest E_N. Interestingly, although the V3 loop epitope targeted by mAb 3074 is present in over 87% of all viruses, it is 82.2% masked, so its E_N is lower than that for mAb 2219. Notably, 50% of the viruses that mAb 3074 is able to neutralize are classified as subtype C viruses, while 70% or more of the viruses neutralized by mAbs 2219, 2557 or 447-52D are classified as subtype B. Thus, neutralization epitopes (in this case, in the V3 loop) have differential patterns of masking and also display distinct patterns of distribution among circulating HIV-1 viruses. Both factors combine to contribute to the practical vaccine value of any single epitope/mAb. Here we have developed a quantitative score for this value. These results have important implications for rational design of vaccines designed to induce neutralizing Abs by revealing epitopes that are minimally masked and maximally reactive with neutralizing Abs.     

Immunological cross-reactivity against a drug mutated HIV-1 protease epitope after DNA multi-CTL epitope construct immunization

Epitopes in HIV polymerase were analyzed by peptide binding to human leukocyte antigen (HLA) A0201 molecules, the most frequent HLA class in the Caucasian population. We found that HIV-1 protease peptides representing both the wild type and anticipated drug resistance variants of the sequence bound well to HLA-A0201. We also found that wild type as well as a double mutated variant of the epitope was strongly immunogenic in HLA-A0201 transgenic mice, either as individual peptides or encoded in DNA multi-CTL epitope constructs. Immunological cross-reactivity between different variants of the peptide could be seen, suggesting that it may be possible to induce a broad immune response by immunizing with drug resistance-mutated epitopes. This may be of advantage for HIV-1 infected patients since such a response may cause a better outcome of an anti-retroviral drug therapy.     

A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176–190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71.     

The effects of preexisting immunity to influenza on responses to influenza vectors in mice

The use of viral vectors as vaccine candidates has shown promise against a number of pathogens. However, preexisting immunity to these vectors is a concern that must be addressed when deciding which viruses are suitable for use. A number of properties, including the existence of antigenically distinct subtypes, make influenza viruses attractive candidates for use as viral vectors. Here, we evaluate the ability of influenza viral vectors containing inserts of foreign pathogens to elicit antibody and CD8⁺ T cell responses against these foreign antigens in the presence of preexisting immunity to influenza virus in mice. Specifically, responses to an H3N1-based vector expressing a 90 amino acid polypeptide derived from the protective antigen (PA) of Bacillus anthracis or an H1N1-based vector containing a CD8⁺ T cell epitope from the glycoprotein (GP) of lymphocytic choriomeningitis virus were evaluated following infections with either homosubtypic or heterosubtypic influenza viruses. We found that mice previously infected with influenza viruses, even those expressing HA and NA proteins of completely different subtypes, were severely compromised in their ability to mount an immune response against the inserted epitopes. This inhibition was demonstrated to be mediated by CD8⁺ T cells, which recognize multiple strains of influenza viruses. These CD8⁺ T cells were further shown to protect mice from a lethal challenge by a heterologous influenza subtype. The implication of these data for the use of influenza virus vectors and influenza vaccination in general are discussed.     

Induction of neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) by immunization with gp41 membrane-proximal external region (MPER) fused with porcine endogenous retrovirus (PERV) p15E fragment

The membrane-proximal external region (MPER) of HIV-1 gp41 is recognized by all three anti-HIV antibodies 2F5, 4E10 and Z13 that were directly derived from AIDS patients and have broader anti-HIV neutralizing activities. Thus, the MPER has been the focus of anti-HIV vaccine design and development. However, it has been unsuccessful to generate anti-HIV neutralizing antibodies targeting this region. One possible reason is that the MPER-containing immunogens have failed to maintain the correct conformation of the MPER present within the HIV-1 viral gp41 protein. The porcine endogenous retrovirus (PERV) p15E protein is structurally similar to HIV-1 gp41, and it was recently reported that the p15E fragment can be expressed in soluble form and was capable of inducing neutralizing antibodies against the epitope within the MPER of PERV p15E. In the present study, we attempted to use the p15E fragment as a carrier and fused the HIV-1 gp41 MPER with the p15E fragment. The vesicular stomatitis virus (VSV) recombinants expressing the fusion proteins (HIV-1 gp41 MPER-p15E) were prepared as primary immunogens, and the soluble fusion protein purified from a baculovirus expression system as booster immunogens. Our results showed that the antisera obtained from immunized rabbits specifically recognized the MPER determinant presented in the gp41 fragment. Importantly, we found that the antisera had neutralizing activities against HIV-1 viruses containing HIV-1 HXB2 and JRFL envelope glycoproteins. These results offer a new strategy for HIV-1 vaccine design and development targeting the gp41 MPER.     

Antibodies elicited by yeast glycoproteins recognize HIV-1 virions and potently neutralize virions with high mannose N-glycans

The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses. Our study demonstrated that the 2G12-reactive TM yeast glycoproteins efficiently bound to recently identified bNAbs including PGT125-130 and PGT135 that recognize high mannose glycan-dependent epitopes. Immunization of rabbits with a single TM yeast glycoprotein (Gp38 or Pst1), when conjugated to a promiscuous T-cell epitope peptide and coadministered with a Toll-like receptor 2 agonist, induced glycan-specific HIV-1 Env cross-reactive antibodies. The immune sera bound to both synthetic mannose oligosaccharides and gp120 proteins from a broad range of HIV-1 strains. The purified antibodies recognized and captured virions that contain both complex- and high mannose-type of N-glycans, and potently neutralized virions from different HIV-1 clades but only when the virions were enforced to retain high mannose N-glycans. This study provides insights into the elicitation of anti-carbohydrate, HIV-1 Env-cross reactive antibodies with a heterologous glycoprotein and may have applications in the design and administration of immunogens that target the viral glycan shield for development of an effective HIV-1 vaccine.     

Immune response to immunostimulatory complexes (ISCOMs) prepared from human immunodeficiency virus type 1 (HIV-1) or the HIV-1 external envelope glycoprotein (gp120)

In mice, immunostimulatory complexes (ISCOMs) prepared from HIV-1 B external envelope glycoprotein (gp120) induced 10-fold higher antibody titres than gp120 emulsified in depot adjuvant, as measured by enzyme-linked immunosorbent assay (ELISA). Rhesus monkeys immunized with gp120 ISCOMs produced precipitating and virus neutralizing antibody titres equivalent to those seen in HIV-infected chimpanzees and humans. After multiple immunizations with HIV-1 B gp120 ISCOMs, a rhesus monkey developed a neutralizing response to the HIV-1 isolates RF and MN, but not to the CC isolate. Antisera from ISCOM-immunized rhesus monkeys recognized gp120 on the membranes of HIV-1 B-infected H9 cells, indicating the preservation of epitope structure in the ISCOMs matrix.     

Bat-transmitted human rabies outbreaks, Brazilian Amazon

We describe 2 bat-transmitted outbreaks in remote, rural areas of Portel and Viseu Municipalities, Pará State, northern Brazil. Central nervous system specimens were taken after patients' deaths and underwent immunofluorescent assay and histopathologic examination for rabies antigens; also, specimens were injected intracerebrally into suckling mice in an attempt to isolate the virus. Strains obtained were antigenically and genetically characterized. Twenty-one persons died due to paralytic rabies in the 2 municipalities. Ten rabies virus strains were isolated from human specimens; 2 other cases were diagnosed by histopathologic examination. Isolates were antigenically characterized as Desmodus rotundus variant 3 (AgV3). DNA sequencing of 6 strains showed that they were genetically close to D. rotundus-related strains isolated in Brazil. The genetic results were similar to those obtained by using monoclonal antibodies and support the conclusion that the isolates studied belong to the same rabies cycle, the virus variants found in the vampire bat D. rotundus.     

Motif-optimized subtype A HIV envelope-based DNA vaccines rapidly elicit neutralizing antibodies when delivered sequentially

HIV-1 infection results in the development of a diverging quasispecies unique to each infected individual. Envelope (Env)-specific neutralizing antibodies (NAbs) typically develop over months to years after infection and initially are limited to the infecting virus. In some subjects, antibody responses develop that neutralize heterologous isolates (HNAbs), a phenomenon termed broadening of the NAb response. Studies of co-crystalized antibodies and proteins have facilitated the identification of some targets of broadly neutralizing monoclonal antibodies (NmAbs) capable of neutralizing many or most heterologous viruses; however, the ontogeny of these antibodies in vivo remains elusive. We hypothesize that Env protein escape variants stimulate broad NAb development in vivo and could generate such NAbs when used as immunogens. Here we test this hypothesis in rabbits using HIV Env vaccines featuring: (1) use of individual quasispecies env variants derived from an HIV-1 subtype A-infected subject exhibiting high levels of NAbs within the first year of infection that increased and broadened with time; (2) motif optimization of envs to enhance in vivo expression of DNA formulated as vaccines; and (3) a combined DNA plus protein boosting regimen. Vaccines consisted of multiple env variants delivered sequentially and a simpler regimen that utilized only the least and most divergent clones. The simpler regimen was as effective as the more complex approach in generating modest HNAbs and was more efficient when modified, motif-optimized DNA was used in combination with trimeric gp140 protein. This is a rationally designed strategy that facilitates future vaccine design by addressing the difficult problem of generating HNAbs to HIV by empirically testing the immunogenicity of naturally occurring quasispecies env variants.     

Immune response in mice inoculated with plasmid DNAs containing multiple-epitopes of foot-and-mouth disease virus

This paper focuses on the development of candidate DNA vaccine encoding antigenic epitopes of type O foot-and-mouth disease virus (FMDV). A series of plasmids encoding different combinations of B cell epitopes and a T cell epitope were constructed and characterized by inoculating BALB/c mice. The specific antibodies were only detectable in the mice inoculated with plasmids encoding the T cell epitope and B cell epitopes from sites 5 and 1, within which site 5 includes residues 135–167 of VP1 and site 1 includes 141–160 region (G–H loop) and carboxyl terminus of VP1. Stronger cellular immune responses were also observed in these mice using T cell proliferation assay.     

A universal anti-HIV-1 Tat epitope vaccine that is fully synthetic and self-adjuvanting

Circulating HIV-1 Tat protein is essential for maintenance of the chronic HIV replication that predicates both HIV transmission and clinical progression to AIDS/death. A synthetic anti-Tat epitope vaccine (TUTI-16) was designed to induce neutralizing antibodies to Tat and, hopefully, provide immunological control of HIV replication. TUTI-16 is composed of (1) a conserved Tat B cell epitope (Tat 4–12), rendered universal by introducing known variant amino acids at variable positions 7, 9 and 12 during solid phase synthesis, (2) a promiscuous T helper sequence and (3) a lipopeptide toll-like receptor 2 (TLR2) agonist. TUTI-16 induced high titer antibodies against all 8 known variants of the Tat epitope.