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An effective prophylactic HIV-1 vaccine is essential in order to contain the HIV/AIDS global pandemic. The discovery of different broadly neutralizing antibodies (bnAbs) in the last decades has enabled the characterization of several minimal epitopes on the HIV envelope (Env) spike, including glycan-dependent fragments. Herein, we provide a brief overview of the progress made on the development of synthetic carbohydrate-based epitope mimics for the elicitation of bnAbs directed to certain regions on Env gp120 protein: the outer domain high-mannose cluster and the variable loops V1V2 and V3. We focus on the design, synthesis and biological evaluation of minimal immunogens and discuss key aspects towards the development of a successful protective vaccine against HIV-1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Despite a very active research in the field of nanomedicine, only a few nano-based drug delivery systems have reached the market. The "death valley" between research and commercialization has been partially attributed to the limited characterization and reproducibility of the nanoformulations. Our group has previously reported the potential of a peptide-based nanovaccine candidate for the prevention of SIV infection in macaques. This vaccine candidate is composed of chitosan/dextran sulfate nanoparticles containing twelve SIV peptide antigens. The aim of this work was to rigorously characterize one of these nanoformulations containing a specific peptide, following a quality-by-design approach. The evaluation of the different quality attributes was performed by several complementary techniques, such as dynamic light scattering, nanoparticle tracking analysis, and electron microscopy for particle size characterization. The inter-batch reproducibility was validated by three independent laboratories. Finally, the long-term stability and scalability of the manufacturing technique were assessed. Overall, these data, together with the in vivo efficacy results obtained in macaques, underline the promise this new vaccine holds with regard to its translation to clinical trials. Graphical abstract.

We generalize the notion of λ-superstrings, presented in a previous paper, to the notion of weighted λ-superstrings. This generalization entails an important improvement in the applications to vaccine designs, as it allows epitopes to be weighted by their immunogenicities. Motivated by these potential applications of constructing short weighted λ-superstrings to vaccine design, we approach this problem in two ways. First, we formalize the problem as a combinatorial optimization problem (in fact, as two polynomially equivalent problems) and develop an integer programming (IP) formulation for solving it optimally. Second, we describe a model that also takes into account good pairwise alignments of the obtained superstring with the input strings, and present a genetic algorithm that solves the problem approximately. We apply both algorithms to a set of 169 strings corresponding to the Nef protein taken from patiens infected with HIV-1. In the IP-based algorithm, we take the epitopes and the estimation of the immunogenicities from databases of experimental epitopes. In the genetic algorithm we take as candidate epitopes all 9-mers present in the 169 strings and estimate their immunogenicities using a public bioinformatics tool. Finally, we used several bioinformatic tools to evaluate the properties of the candidates generated by our method, which indicated that we can score high immunogenic λ-superstrings that at the same time present similar conformations to the Nef virus proteins., Competing Interests: The authors have declared that no competing interests exist.

We have demonstrated that a liposomal array of well-ordered trimers enhances B cell activation, germinal center formation, and the elicitation of tier-2 autologous neutralizing antibodies. Previously, we coupled well-ordered cleavage-independent NFL trimers via their C-terminal polyhistidine tails to nickel lipids integrated into the lipid bilayer. Despite favorable in vivo effects, concern remained over the potentially longer-term in vivo instability of noncovalent linkage of the trimers to the liposomes. Accordingly, we tested both cobalt coupling and covalent linkage of the trimers to the liposomes by reengineering the polyhistidine tail to include a free cysteine on each protomer of model BG505 NFL trimers to allow covalent linkage. Both cobalt and cysteine coupling resulted in a high-density array of NFL trimers that was stable in both 20% mouse serum and 100 mM EDTA, whereas the nickel-conjugated trimers were not stable under these conditions. Binding analysis and calcium flux with anti-Env-specific B cells confirmed that the trimers maintained conformational integrity following coupling. Following immunization of mice, serologic analysis demonstrated that the covalently coupled trimers elicited Env-directed antibodies in a manner statistically significantly improved compared to soluble trimers and nickel-conjugated trimers. Importantly, the covalent coupling not only enhanced gp120-directed responses compared to soluble trimers, it also completely eliminated antibodies directed to the C-terminal His tag located at the "bottom" of the spike. In contrast, soluble and noncovalent formats efficiently elicited anti-His tag antibodies. These data indicate that covalent linkage of well-ordered trimers to liposomes in high-density array displays multiple advantages in vitro and in vivo IMPORTANCE Enveloped viruses typically encode a surface-bound glycoprotein that mediates viral entry into host cells and is a primary target for vaccine design. Liposomes with modified lipid head groups have a unique feature of capturing and displaying antigens on their surfaces, mimicking the native pathogens. Our first-generation nickel-based liposomes captured HIV-1 Env glycoprotein trimers via a noncovalent linkage with improved efficacy over soluble glycoprotein in activating germinal center B cells and eliciting tier-2 autologous neutralizing antibodies. In this study, we report the development of second-generation cobalt- and maleimide-based liposomes that have improved in vitro stability over nickel-based liposomes. In particular, the maleimide liposomes captured HIV-1 Env trimers via a more stable covalent bond, resulting in enhanced germinal center B cell responses that generated higher antibody titers than the soluble trimers and liposome-bearing trimers via noncovalent linkages. We further demonstrate that covalent coupling prevents release of the trimers prior to recognition by B cells and masks a nonneutralizing determinant located at the bottom of the trimer., (Copyright © 2017 American Society for Microbiology.)

To date, several antigens based on the amino-terminal leucine/isoleucine heptad repeat (NHR) region of an HIV-1 envelope protein gp41 and fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of gp41 have been reported. We have developed a synthetic antigen targeting the membrane-fusion mechanism of HIV-1. This uses a template designed with C3-symmetric linkers and mimics the trimeric form of the NHR-derived peptide N36. The antiserum obtained by immunization of the N36 trimeric antigen binds preferentially to the N36 trimer and blocks HIV-1 infection effectively, compared with the antiserum obtained by immunization of the N36 monomer. Using another template designed with different C3-symmetric linkers, we have also developed a synthetic peptide mimicking the trimeric form of the CHR-derived peptide C34, with ∼100 times the inhibitory activity against the HIV-1 fusion mechanism than that of the monomer C34 peptide. A dimeric derivative of C34 has potent inhibitory activity at almost the same levels as this C34 trimer mimic, suggesting that presence of a dimeric form of C34 is structurally critical for fusion inhibitors. As examples of rising mid-size drugs, this review describes an effective strategy for the design of HIV vaccines and fusion inhibitors based on a relationship with the native structure of proteins involved in HIV fusion mechanisms. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 622-628, 2016., (© 2015 Wiley Periodicals, Inc.)

Knowledge of the sequences and structures of proteins from pathogenic microorganisms has been put to great use in the field of protein chemistry for the development of peptide-based vaccines. These vaccine constructs include chemically tailored, shorter peptidic fragments that can induce high immunogenicity, thus shunning the allergenic and nonimmunogenic part of the antigens. Based on this concept, several different chemistries have been pursued to obtain novel platforms onto which antigenic epitopes can be tethered, with the aim to achieve a higher antibody response. In this regard, here we attempt to summarize the chemical strategies developed for the presentation of peptide epitopes., (© 2015 Elsevier Inc. All rights reserved.)

The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We report herein a unified convergent strategy for the rapid production of bi-, tri-, and tetra-antennary complex type N-glycans with and without terminal N-acetylneuraminic acid residues connected via the α-2,6 or α-2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic steps through the use of shared intermediates to simplify the complicated procedures associated with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides were compiled to create a glycan array for the profiling of HIV-1 broadly neutralizing antibodies PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16, we identified potential natural and unnatural glycan ligands, which may facilitate the design of carbohydrate-based immunogens and hasten the HIV vaccine development.

A major goal in HIV-1 vaccine research is to develop an immunogen that can elicit broadly neutralizing antibodies that efficiently neutralize a wide range of the HIV-1 subtypes. Using biopanning procedure we have selected linear peptide VGAFGSFYRLSVLQS mimicking the structure of discontinuous binding sites of broadly neutralizing antibodies 2G12 from phage peptide library. As a protein carrier, we used the earlier designed artificial polyepitope immunogen named TBI (T- and B-cell immunogen), which comprises B-cell and T-helper epitopes from the HIV-1 Env and Gag proteins. On the base of selected peptide mimotope VGAFGSFYRLSVLQS the artificial protein TBI-2g12 was constructed and its immunogenic properties was investigated. It was shown that the TBI-2g12 as well as the original TBI induces antibodies that recognize HIV-1 proteins and TBI protein using ELISA and immunoblotting. However only anti-TBI-2g12 serum recognized the synthetic peptide mimotope VGAFGSFYRLSVLQS, whereas the antibodies against original TBI don't recognize it. The neutralization assay demonstrated that serum antibodies of the mice immunized with TBI-2g12 possess virus neutralizing activity. The addition of selected peptide leads to inhibition neutralizing activity of anti- TBI-2g12 serum. We conclude from these results that immunogen TBI-2g12 containing the selected peptide VGAFGSFYRLSVLQS elicits HIV-1 neutralizing antibodies during immunization. Our data suggest that this immunogen may be useful in designing effective HIV-vaccine candidates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M) and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application.

The short multiepitopic synthetic peptides from the sequences of hypervariable area of V3-loope of gp120 of HIV don't induce anti-peptides antibodies production in mice themselves. We prepared the potent immunogen by noncovalent conjugations of the multitude peptides with pure peptidoglycans from cell wall of Salmonella typhi. The sera from immunized mice have the anti-peptides antibody titers (3-5) x 10(5) in ELISA, as high as Freund's adjuvant is of use.

Acquired immunodeficiency syndrome and tuberculosis (TB) are two of the world's most devastating diseases. The first vaccine the majority of infants born in Africa receive is Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a prevention against TB. BCG protects against disseminated disease in the first 10 years of life, but provides a variable protection against pulmonary TB and enhancing boost delivered by recombinant modified vaccinia virus Ankara (rMVA) expressing antigen 85A (Ag85A) of M. tuberculosis is currently in phase IIb evaluation in African neonates. If the newborn's mother is positive for human immunodeficiency virus type 1 (HIV-1), the baby is at high risk of acquiring HIV-1 through breastfeeding. We suggested that a vaccination consisting of recombinant BCG expressing HIV-1 immunogen administered at birth followed by a boost with rMVA sharing the same immunogen could serve as a strategy for prevention of mother-to-child transmission of HIV-1 and rMVA expressing an African HIV-1-derived immunogen HIVA is currently in phase I trials in African neonates. Here, we aim to develop a dual neonate vaccine platform against HIV-1 and TB consisting of BCG.HIVA administered at birth followed by a boost with MVA.HIVA.85A. Thus, mMVA.HIVA.85A and sMVA.HIVA.85A vaccines were constructed, in which the transgene transcription is driven by either modified H5 or short synthetic promoters, respectively, and tested for immunogenicity alone and in combination with BCG.HIVA(222). mMVA.HIVA.85A was produced markerless and thus suitable for clinical manufacture. While sMVA.HIVA.85A expressed higher levels of the immunogens, it was less immunogenic than mMVA.HIVA.85A in BALB/c mice. A BCG.HIVA(222)-mMVA.HIVA.85A prime-boost regimen induced robust T cell responses to both HIV-1 and M. tuberculosis. Therefore, proof-of-principle for a dual anti-HIV-1/M. tuberculosis infant vaccine platform is established. Induction of immune responses against these pathogens soon after birth is highly desirable and may provide a basis for lifetime protection maintained by boosts later in life.

A synthetic antigen targeting membrane-fusion mechanism of HIV-1 has a newly designed template with C3-symmetric linkers mimicking N36 trimeric form. The antiserum produced by immunization of the N36 trimeric form antigen showed structural preference in binding to N36 trimer and stronger inhibitory activity against HIV-1 infection than the N36 monomer. Our results suggest an effective strategy of HIV vaccine design based on a relationship to the native structure of proteins involved in HIV fusion mechanisms.

The V3 region of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) is a potential target for an anti-HIV-1 vaccine. Peptides corresponding to V3 form three variations of a beta-hairpin conformation when bound to anti-V3 HIV-1 neutralizing antibodies. The conformation of a V3(IIIB) peptide bound to the 0.5beta antibody, generated against an X4 gp120, has been postulated to represent the V3 conformation of X4 viruses while the conformations of a V3(MN) and a V3(CONSENSUS) peptide bound to the 447-52D human monoclonal antibody were postulated to represent the R5A and R5B V3 conformations of R5 viruses, respectively. To constrain the conformation of synthetic V3 peptides to these X4, R5A, and R5B conformations, we formed disulfide bonds between Cys residues whose location in a peptide template representing the entire V3(CONSENSUS) epitope recognized by the broadly neutralizing 447-52D antibody was changed systematically. In a previous study [Mor, A., et al. (2009) Biochemistry 48, 3288-3303] we showed that these constrained peptides adopted conformations resembling the three antibody-bound V3 conformations according to the location of the disulfide bonds. Here we show that these constrained peptides, with the exception of peptides in which the disulfide bond flanks the GPGR segment, retain high-affinity binding to the 447-52D antibody. Compared with peptides designed to mimic the X4 conformation, peptides designed to mimic either the R5A or R5B conformation had higher affinity to 447-52D. It is possible that constrained peptides which mimic the R5A and R5B conformations of the V3 and retain high-affinity binding to 447-52D are good candidates for eliciting a broad neutralizing antibody response similar to that of 447-52D.

One of the major obstacles in the design of an effective vaccine against HIV-1 is its antigenic variation, which results in viral escape from the immune system. Through a bioinformatics approach, we developed an innovative multivalent HIV-1 vaccine comprised of a pool of 176 lipidated and nonlipidated peptides representing variable regions of Env and Gag proteins. The potency and breadth of the candidate vaccine against a panel of HIV-1 subtypes was evaluated in nonhuman primate (cynomolgus macaques) and humanized mouse (HLA-A2.1) models. The results demonstrate strong immunogenicity with both breadth (humoral and cellular immunity) and depth (immune recognition of widely divergent viral sequences) against heterologous HIV-1 subtypes A-F.

Human immunodeficiency virus type 1 (HIV-1) requires a chemokine receptor (CCR5 or CXCR4) as a coreceptor not only for initiate viral entry but also protecting highly conserved neutralization epitopes from the attack of neutralizing antibodies. Over the past decade, many studies have provided new insights into the HIV entry mechanism and have focused on developing an effective vaccine strategy. However, to date, no vaccine that can provide protection from HIV-1 infection has been developed. One reason for the disappointing results has been the inability of current vaccine candidates to elicit a broadly reactive immunity to viral proteins such as the envelope (env) protein. Here, we propose that chemokine receptors are attractive targets of vaccine development because their structures are highly conserved and that our synthetic cycloimmunogens can mimic conformational-specific epitopes of undecapeptidyl arches (UPAs: R(168)-C(178) in CCR5, N(176)-C(186) in CXCR4) and be useful for HIV-1 novel vaccine development.

The development of an HIV/AIDS vaccine requires consideration of the large diversity of viral isolates. In 2005, there were 5 million new cases of HIV infection and over 4 million deaths due to AIDS. An HIV vaccine is needed to prevent the spread of this virus. One of the greatest challenges to developing a preventative HIV vaccine is the diversity of HIV-1 isolates. Env sequences can differ by as much as 35% between isolates from different clades and by as much as 10% within a clade. Two main strategies to address viral diversity for HIV vaccine development are the use of polymeric- or centralized-based immunogens. Polymeric-based vaccines, which have been used for polio and pneumococcus vaccines, use components from a range of viral isolates to increase the breadth of immune recognition. Centralized sequences decrease the sequence diversity by encoding the most common amino acid at each position from a diverse pool of viral isolates. These sequences are derived using the consensus, center-of-the-tree, or ancestral methods. The use of polyvalent- and centralized-based vaccines induce broadly reactive immune responses, however it is unclear whether the use of these sequences will increase protection against diverse HIV-1 infection. This review will summarize the current uses of polyvalent and centralized vaccines to increase immune breadth that may determine future directions for HIV-1 vaccine development.

An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will likely require the elicitation of broadly neutralizing antibodies as well as cellular responses. The HIV exterior envelope glycoprotein trimers, gp120, and the transmembrane glycoprotein, gp41, mediate entry and are the sole viral targets for neutralizing antibodies. However, as subunit immunogens the envelope glycoproteins do not efficiently elicit antibodies capable of neutralizing the extremely diverse array of viruses circulating in the human population. The preponderance of data suggest that inefficient generation of broadly neutralizing antibodies is due to naturally evolved mechanisms of immune evasion inherent in the unmodified HIV envelope glycoproteins. Because the established modes of anti-viral vaccine development, live-attenuation and virus inactivation have not yet been successful for HIV, we and others have focused on subunit vaccine design. In this review, we describe current approaches of rational modification of the envelope glycoproteins based upon structure, antigenicity, biochemistry and biophysics to alter the properties of the envelope glycoproteins such that, as subunit immunogens, they now better elicit broadly neutralizing antibodies. The application of structure-assisted, rational subunit vaccine design may be a general paradigm for future efforts to develop vaccines against emerging human pathogens.

The need for an affordable, safe and effective HIV vaccine has never been greater. As the immunogenicity of all the vaccine vectors being evaluated currently in human populations is limited, novel vaccine strategies are needed to stimulate the innate immune system, to generate high levels of neutralizing antibodies and to induce strong cell-mediated and mucosal immunity. There is strong evidence for a role for cytotoxic T lymphocytes in the containment of HIV replication. Several vaccine approaches have been tested to elicit anti-HIV cytotoxic T-lymphocyte responses. One promising approach is Bacillus Calmette-Guérin (BCG) as a bacterial live recombinant vaccine vehicle. BCG has a long record of safety in humans and is able to induce long-lasting immunity. In this review, we describe the limitations and challenges of developing a recombinant BCG-based HIV vaccine. We also emphasize possible approaches for overcoming the plasmid instability in vivo and the low levels of gene expression and immunogenicity induction. Today, projects all over the world are focused on the development of an AIDS vaccine. Overcoming the remaining scientific, logistical and financial hurdles to the development of an effective HIV vaccine will require real imagination and firm commitment from all stakeholders.

Natural miniproteins (e.g., animal toxins, protease inhibitors, defensins) can express specific and powerful biological activities by using a stable and minimal (<80 amino acids) structural motif. Artificial activities have been designed on these miniscaffolds by transferring previously identified protein active sites into regions structurally compatible with the site and permissive for sequence mutations. These newly designed miniproteins, presenting a specific and high activity within a small size and well-defined three-dimensional structure, represent novel tools in biology, biotechnology, and medical sciences, and are also useful intermediates to develop new therapeutic agents. The different steps used to design and characterize new bioactive miniproteins are here described in detail. Two successful examples are here reported. The first one is a metal-binding miniprotein (MBP, 37 residues), which possesses a metal specificity resembling that of natural carbonic anhydrase; the second is a CD4 mimic (CD4M33, 27 residues), which is a powerful inhibitor of HIV-1 entry but also a fully functional substitute of the human receptor CD4 and, hence, a potential component of an AIDS vaccine.

Induction of high levels of broadly reactive cytotoxic T lymphocytes (CTL) remains a promising approach for an effective HIV-1 vaccine. We have developed a novel genetic-based vaccine strategy that encodes consensus overlapping peptide sets from all HIV-1 proteins scrambled together. This synthetic scrambled antigen vaccine (SAVINE) strategy has significant advantages, e.g. capacity to encode more antigens safely and is very flexible compared to traditional isolate-based strategies. The SAVINE vaccine strategy is clearly immunogenic, being able to restimulate a range of human HIV-1 specific responses in vitro and induce HIV-1 specific immunity in vivo in mice. Interestingly, different in vivo delivery strategies affected the resulting immunity and immunodominance pattern in mice. This platform strategy could be used for other infections and cancers where T cell responses are important for protection.

Synthetic V3 loop peptides hold numerous benefits for the development of HIV vaccines and in immunodiagnosis; however, their use is limited due to the extensive antigenic variability of this region. The effectiveness of a potential HIV vaccine component, which accounts for V3 loop variability, is evaluated here. A branched peptide construct representing multiple sequences and allowing 1.8 x 10(16) possible permutations was developed to mimic circulating HIV-1 subtype C, V3 loops. The construct was found to be immunogenic, able to induce neutralizing antibodies and sensitive to HIV-1 subtype B/C and HIV-2. This alternative antigen format also incorporates conformational epitopes.

VIRxSYS is developing VRX-496, a lentiviral HIV-based vector encoding anti-HIV antisense envelope sequences, as a potential gene therapy for HIV infection. In July 2003, VIRxSYS undertook the initial dosing of an HIV-positive patient in a phase I/IIa trial.

Vaccines against highly variable pathogens should elicite antibodies to a huge number of clinical isolates. For this purpose, new strategies to overcome the variability are needed. We have previously reported a useful method to conjugate multiple antigen peptides (MAPs) to carrier proteins. Also, we have suggested that these conjugates might enhance cross-reactivity in comparison to other synthetic structures. In this work, MAPs were synthesized and their respective conjugates to HBsAg were obtained. Two peptides from the V3 loop of HIV-1 were included in the MAPs as B cell epitopes because of their variability. Groups of mice were immunized and the immunogenicity and the level of cross-reaction to a panel of five heterologous V3 peptides were studied. Our results show that sera from mice immunized with MAPs coupled to HBsAg recognize a higher number of heterologous peptides (P < 0.05). This behavior was related neither to the immunogenicity nor the antigenicity of the synthetic structures. These results have important implications for the choice of better immunogens against variable epitopes.

Different strategies have been used to increase the immunogenicity of an antigenic HIV peptide as a vaccine candidate. The selected B-cell epitope comprises 15 amino acids (317-331) of the V3 region of HIV-1, JY1 isolate (subtype D), in tandem with a T-helper epitope corresponding to the 830-844 region of tetanus toxoid. Several presentations, including oligomerization, multiple antigenic peptide dendrimers, and conjugation to dextran beads or to other macromolecular carriers, have been synthesized and evaluated. Murine sera from the different presentations of the V3 epitope have been compared with regard to antibody titers and cross-reactivity with heterologous HIV subtypes. The dendrimer version of the peptide conjugated to HBsAg protein was a better immunogen than the dendrimer alone and showed a higher immunogenicity than other multimeric presentations or than the peptide alone conjugated to dextran. The dendrimer version, either alone or conjugated to HBSAg, enhanced cross-reactivity toward heterologous V3 sequences relative to monomeric peptide. In addition, fine epitope mapping of the entire JY1 sequence by sera from the different immunization groups was performed by the spot synthesis technique. Results showed that the amino acids involved in molecular recognition were LXQXXY or LXQXLY, with particularly strong recognition of the C-terminal region LGQALY. However, cross-reactivity toward the heterologous sequences did not completely correlate with recognition of particular amino acids in the primary sequences. These results can find application in the development of HIV vaccine candidates.

Several studies have suggested that HIV-1 p17 matrix protein may play an important role in AIDS pathogenesis, since anti-p17 antibodies represent a serological marker of disease progression during HIV-1 infection both in adults and children. Moreover, it has been recently reported that the viral protein is capable of significantly increasing the proliferation of preactivated T lymphocytes and the release of proinflammatory cytokines. Recombinant HIV-1 p17 also has induced an increased rate of HIV-1 replication in vitro. All p17 biological activities are exerted after its binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH(2)-terminal region of the viral protein. Immunization of C57BL/6 mice with a 20 amino acid synthetic peptide representative of the HIV-1 p17 functional region (AT20) coupled to the carrier protein keyhole limpet hemocyanin (KLH) and given in Freund's incomplete adjuvant, resulted in the development of p17-neutralizing antibodies capable of blocking p17/p17 receptor interaction, and consequently, all biological activities of the viral protein. Moreover, it was possible to skew the humoral response induced by priming mice with AT20-KLH toward cell-mediated immune responses, boosting animals with p17. Our findings may provide a new strategy to develop a synthetic AIDS vaccine based on a potentially effective and safe subunit vaccine against the HIV-1 cytokine-like matrix protein p17. Preclinical immunogenicity data for AT20-KLH provide the basis for evaluation of the peptide-based vaccine, alone and in combination with p17 or p17 DNA vaccines, in Phase I clinical trials.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-gamma ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.

Despite the remarkable advances that have been made in the last 20 years regarding the molecular virology, pathogenesis and epidemiology of HIV, the development of an effective HIV vaccine remains an elusive goal. The major reason for this is that we have not determined a correlate of immunity. The various explantations for this include integration of the virus into the host cell genome, infection of long-lived immune cells, HIV genetic diversity (especially in its envelope), persistent high viral replication releasing up to 10 billion viral particles per day and/or production of immunosuppressive products or proteins. However, there is evidence that the host can be protected: some highly exposed persons have remained uninfected; the relatively low incidence of mother to child (fetus) transmission; the initial effective immune response that significantly, if temporally, reduces viral loads; some infected persons are long-term non-progressors; experimental vaccines and passive immunisation have proven effective in experimental animals; and finally, successful vaccine development against other viral infections. At this time, the experimental vaccine pipeline is quite robust and ranges from HIV proteins (although the first such vaccine, recombinant gp120 made on Chinese hamster ovary cells, failed to protect volunteer men having sex with men [MSM]) to DNA vaccines and various novel delivery strategies. Perhaps the greatest impediment is the requirement to test these experimental vaccines in resource-poor developing countries that, at present, lack the necessary infrastructure for performing large, long-term, scientifically valid studies.

One of the major problems in the development of successful recombinant vaccines against human immunodeficiency virus (HIV) is that of correct identification of a safe and effective vaccine delivery system with which to induce protective immunity using soluble protein antigens. An original method for constructing artificial immunogens in the form of spherical particles with yeast dsRNA in the center and hybrid proteins exposing epitopes of an infectious agent on the surface is reported. The dsRNA and the proteins were linked with spermidine-polyglucin-glutathione conjugates. Particles exposing HIV-1 epitopes were constructed, and their immunogenicity tested.

A class of synthetic peptide immunogens for the cell surface HIV receptor complex has been developed to elicit antibodies that block viral entry by inhibiting gp120-CD4 interaction. These peptides extend our HIV receptor-directed approach from passive immunotherapy with mAb B4 (Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 10367) to active immunization by a synthetic peptide-based vaccine. A peptide site from CD4 was identified as a B cell epitope capable of mimicking a susceptible site on the HIV receptor complex, and then rendered immunogenic. An effective target antigenic site (B cell epitope) for the cell surface HIV receptor complex was selected by epitope mapping from among diverse CD4 and chemokine receptor peptides. It is a cyclized sequence modified from the CDR2-like domain of CD4 (AA 39-66), that was predicted to produce steric hindrance of the discontinuous recognition site of mAb B4. The immunogenicity of the targeted epitope was augmented by tandem combination with promiscuous T helper cell epitopes (Th). The antibody response to this class of immunogens attained sufficient concentrations and affinities of the correct specificity to block the interactions of HIV env glycoprotein with the cellular receptor, and prevent infection. The polyclonal antibodies generated against these fusion constructs in multiple animal species neutralized a broad array of HIV-1 primary isolates from clades A to E. Despite eliciting antibodies to the key CD4 immunomodulatory molecule, the site-specific and chemically defined immunogens displayed no overt immunotoxicity in baboons and have potential for the immunotherapy and immunoprophylaxis of HIV infection.

The Immune Response Corp (IRC) is developing Remune, a potential HIV therapeutic vaccine. Remune is based on the Salk Immunogen, which is derived from an HIV isolate which has been inactivated by chemical depletion of glycoprotein 120 (gp120). Preliminary data suggested that Remune, in combination with antiviral drug therapy, results in undetectable levels of HIV. Phase III trials commenced in May 1997 and it was initially expected that registration filings would be made in 1999. However, following interim analysis of the 2500-patient, multicenter, double-blind, pivotal phase III study (study 806) in May 1999, an independent panel recommended concluding the clinical endpoint trial and IRC and licensee, Agouron, decided to pursue alternative regulatory strategies, including initiating two additional phase III surrogate marker trials. Despite this, Agouron gave IRC notice of termination of its continued development in July 2001. In August 2001, IRC informed Agouron that, due to the total number of endpoints to date falling short of that previously assumed by Agouron, it did not intend to continue Agouron's Study 202 of Remune. In July 2001, licensee Trinity Medical Group filed an NDA with the governing health authorities in Thailand for Remune. The Thai FDA certified Immune Response's Remune manufacturing facility as being in compliance with GMP standards, following an on site inspection by Thai officials in November 2001 that was performed as a requirement of Trinity's Thai NDA. As a result of this certification, Trinity expected that a "timely determination" could be made by the Thai FDA. Rhĵne-Poulenc Rorer discontinued its part in the development of Remune, with all manufacturing, marketing and distribution rights reverting to IRC. After Agouron returned rights to Remune in July 2001, IRC heldfull rights in the US, Europe and Japan, while collaborating with its partners Trinity Medical Group and Roemmers Laboratory in the Southeast Asian and Latin American markets, respectively. In June 1998, Merrill Lynch had expected an FDA filing for Remune by the end of 1999, and in May 1999 Agouron was hoping to file an NDA for the use of Remune against AIDS during 2001. Analysts at Lehman Brothers predicted in September 2001, that the product would be launched onto the market with sales of US$150 million in 2004.

Recent advances in HIV vaccine development include initiation of the first efficacy trials and substantial expansion of the preclinical pipeline. Several preclinical candidate vaccines have induced strong cellular immune responses and provided impressive protection against AIDS in non-human primate models; however, candidates that induce broadly neutralizing antibodies remain elusive.

Novel conformation-specific antibodies were raised against a cyclic chimeric dodecapeptidyl multiple antigen peptide (cCD-MAP) constructed with a spacer-armed Gly-Asp dipeptide and two pentapeptides (S(169)-Q(170)-K(171)-E(172)-G(173) of CCR5 and E(179)-A(180)-D(181)-D(182)-R(183) of CXCR4) which are components of the undecapeptidyl arch (UPA: from R(168) to C(178) in CCR5, from N(176) to C(186) in CXCR4) of extracellular loop 2 (ECL2) in chemokine receptors (CCR5 and CXCR4). Of the antibodies raised, one monoclonal antibody, CPMAb-I (IgMkappa), reacted with cCD-MAP, but not with the linear chimeric dodecapeptide-MAP. The antibody reacted with the cells separately expressing CCR5 or CXCR4, but not with those not expressing the coreceptors. Moreover, the antibody markedly suppressed infection by X4, R5, or R5X4 virus in a dose-dependent manner in a new phenotypic assay for drug susceptibility of HIV-1 using CCR5-expressing Hela/CD4(+) cell clone 1-10 (MAGIC-5). Moreover, CPMAb-I interfered with LAV-1(BRU) infection (m.o.i. = 0.01) of Molt4#8 cells cocultured with CPMAb-I-producing hybridoma in the transwell, and significantly interfered with neither chemotaxis nor calcium influx induced with stromal cell-derived factor 1 alpha (SDF-1alpha). Thus, the antibody raised against the cCD-MAP provides powerful protection or defense against HIV-1 infection. We therefore propose the cCD-MAP or its derivative immunogen as a novel candidate for an HIV-1 coreceptor-based self-defense vaccine., (Copyright 2001 Academic Press.)

Immunostimulatory DNA sequences (ISS, also known as CpG motifs) are pathogen-associated molecular patterns that are potent stimulators of innate immunity. We tested the ability of ISS to act as an immunostimulatory pathogen-associated molecular pattern in a model HIV vaccine using gp120 envelope protein as the Ag. Mice immunized with gp120 and ISS, or a gp120:ISS conjugate, developed gp120-specific immune responses which included: 1) Ab production; 2) a Th1-biased cytokine response; 3) the secretion of beta-chemokines, which are known to inhibit the use of the CCR5 coreceptor by HIV; 4) CTL activity; 5) mucosal immune responses; and 6) CD8 T cell responses that were independent of CD4 T cell help. Based on these results, ISS-based immunization holds promise for the development of an effective preventive and therapeutic HIV vaccine.

Since the mid-1990s, southern African countries have been experiencing an expansion of human immunodeficiency virus type 1 (HIV-1) infection caused by HIV-1 subtype C. To facilitate the design of an HLA-based HIV vaccine, we studied the distribution of the HLA class I antigen specificities in Botswana, a southern African country with a high prevalence of HIV infection. Botswana's highly efficient health care system and its central geographical location within southern Africa suggests that it might be an appropriate candidate site for future trials of an HLA-based HIV vaccine. Specificities of HLA class I genes have been investigated in DNA samples obtained from 161 persons of Botswana origin by polymerase chain reaction (PCR) with sequence-specific primers. We identified 4 HLA-A, 7 HLA-B, and 5 HLA-C specificities that were observed at high frequencies in the Botswana population: A30, A02, A23, A68, B58, B72, B42, B8, B18, B44, B45, Cw7, Cw2, Cw17, Cw6, and Cw4. HLA-A30, A02, A23, A68, B58, Cw2, Cw4, Cw6, Cw7, and Cw17 were observed at frequencies of more than 10%. The frequency of HLA-A30 was 27.3%. HLA-B58 (17.9%) was the most frequent generic HLA-B type. Other frequent antigen specificities detected for the HLA-B were B72 (9.6%), B42 (9.3%), B8 (7.4%), B18 (7.4%), B44 (7.4%), and B45 (6.4%). Analysis of haplotype frequencies revealed that haplotypes HLA-A30/HLA-B58 (6.7%), A30/B42 (6.1%), A30/B8 (4.1%), A30/B45 (3.2%), and A23/B58 (2.5%) were the most frequent among two-locus haplotypes. The comparison of HIV-positive patients and noninfected controls for HLA class I specificities confirmed the previously described association of A2/A6802 supertype with resistance to HIV. Our study suggested an increased resistance to HIV infection associated with A68 rather than A2. We also found that the generic HLA-B58 type was associated with increased susceptibility to HIV infection. Our findings suggest that the design of an HLA-based HIV vaccine that includes multiple CTL epitopes restricted by identified common HLA class I specificities might target up to 97.5% of the population in Botswana. The results of this study extend the HLA map to a southern African country that has high rates of HIV and also provide a database for the design of an HLA-based HIV vaccine.

We examined the interplay between cytokines and adjuvants to optimize the induction of CTL by a mucosal HIV peptide vaccine. We show synergy between IL-12 and GM-CSF when administered together with the HIV peptide PCLUS3-18IIIB and cholera toxin (CT) in the induction of CTL activity and protection against mucosal viral transmission. Further, we examine the efficacy of mutant Escherichia coli labile toxin, LT(R192G), as a less toxic adjuvant than CT. LT(R192G) was as effective as or more effective than CT at inducing a mucosal CTL response. Moreover, LT(R192G) was as effective without IL-12 as CT was when combined with IL-12, and the response elicited by LT(R192G) with the vaccine was not further enhanced by the addition of IL-12. GM-CSF synergized with LT(R192G) without exogenous IL-12. Therefore, LT(R192G) may induce a more favorable cytokine response by not inhibiting IL-12 production. In particular, less IL-4 is made after LT(R192G) than CT immunization, and the response is less susceptible to anti-IL-12 inhibition. Thus, the choice of mucosal adjuvant affects the cytokine environment, and the mucosal response and protection can be enhanced by manipulating the cytokine environment with synergistic cytokine combinations incorporated in the vaccine.

The generation of neutralizing antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native protein. Consequently, approaches are needed for developing conformational mimics of protein neutralization sites. We replace putative main-chain hydrogen bonds (NH --> O=CRNH) with a hydrazone link (N-N=CH-CH(2)CH(2)) and scan constrained peptides for fit with neutralizing monoclonal antibodies (MAbs). To explore this approach, a V3 MAb 58.2 that potently neutralizes T-cell lab-adapted HIV-1(MN) was used to identify a cyclic peptide, [JHIGPGR(Aib)F(D-Ala)GZ]G-NH(2) (loop 5), that binds with >1000-fold higher affinity than the unconstrained peptide. NMR structural studies suggested that loop 5 stabilized beta-turns at GPGR and R(Aib)F(D-Ala) in aqueous solvent implying considerable conformational mimicry of a Fab 58.2 bound V3 peptide determined by X-ray crystallography [Stanfield, R. L. et al. (1999) Structure 142, 131-142]. Rabbit polyclonal antibodies (PAbs) generated to loop 5 but not to the corresponding uncyclized peptide bound the HIV-1(MN) envelope glycoprotein, gp120. When individual rabbit antisera were scanned with linear and cyclic peptides, further animal-to-animal differences in antibody populations were characterized. Loop 5 PAbs that most closely mimicked MAb 58.2 neutralized HIV-1(MN) with similar potency. These results demonstrate the remarkable effect that conformation can have on peptide affinity and immunogenicity and identify an approach that can be used to achieve these results. The implications for synthetic vaccine and HIV-1 vaccine research are discussed.

Variability of the major antigenic sites of the envelope glycoprotein of HIV-1 constitutes a major problem in the formulation of effective vaccines. We have prepared a synthetic peptide vaccine that represents the major hypervariable epitopes (V1 through V5) of the clade B HIV-1 envelope glycoprotein (gp120). We refer to this preparation as variable epitope immunogen or VEI vaccine. This construct takes into consideration the type and frequency of amino acid substitutions found at each epitope during the evolution of the virus in individual patients and in the target population. Immunization of mice, rabbits, and rhesus macaques with the VEI vaccine resulted in the induction of long-lasting, high-titered HIV-1 antibodies, including antibodies that neutralize primary isolates. We also documented lymphocyte proliferative responses to the VEI vaccine, its individual components, analogs, and subtype-specific peptides representing the major hypervariable regions of HIV-1 gp120. Delayed-type hypersensitivity responses to these antigens were also demonstrated in mice. Our results show that this vaccine is highly immunogenic and safe in animals. Our data suggest that this formulation could become an important component of combination vaccine approaches against HIV-1 and other antigenically variable pathogens.

The use of synthetic peptide antigens in human prophylaxis still suffers from the very important problem of finding suitable carriers devoid of side effects. A desirable carrier for use in humans would be poorly immunogenic by itself, yet it would enhance the immune response to the peptide antigen. In the study reported herein, we examined the role of polytuftsin (TKPR40), a synthetic polymer of the natural immunomodulator tuftsin, as a carrier for synthetic peptides of HIV derived from the gp41 and gp120 proteins. Chimeric immunogens were constructed by chemical linkage between synthetic peptides of HIV and polytuftsin. These were employed for immunization of mice of different MHC haplotypes, and the humoral and cellular immune responses developed against the peptides were assessed by measuring total IgG, IgG, subclasses, T-cell proliferation, and in vitro cytokine release. A significantly stronger immune response was observed in mice immunized with the peptide-polytuftsin conjugates than in mice receiving the peptide dimers (peptide-peptide). Peptide-polytuftsin conjugates induced IgG2a and IgG2b isotype switching after both primary and secondary immunization. In addition, there was a positive correlation between the amounts of cytokines and the shift in the IgG isotypes. These data suggest that the use of polytuftsin as a carrier may increase the immune response against poorly immunogenic synthetic peptides.

Advances have been made in the development of vaccines based on synthetic peptides and polypeptides representing tumor-associated antigens and protective epitopes of viruses and parasites. Advances within the past year include the design of vaccines based on artificial proteins, for example multiantigen peptides, branched polypeptides, fusion and recombinant peptides, as well as single T cell epitopes and tumor antigen peptides. Although peptide vaccines are not in use as yet, their potential is being explored.

Nucleic acid or DNA immunization represents a novel approach to both vaccine and immune therapeutic development. DNA vaccination induces antigen-specific cellular and humoral immune responses through the delivery of non-replicating transcription units which drive the synthesis of specific foreign proteins within the inoculated host. We have previously reported on the potential use of DNA immunization as a novel vaccine strategy for HIV-1. We found that both antigen-specific cellular and humoral immune responses could be induced in vivo with various DNA vaccine constructs against different antigenic targets within HIV-1. In order to enhance the DNA vaccine's ability to elicit cell-mediated immune responses, we co-delivered plasmids encoding costimulatory molecule B7 and interleukin-12 genes with DNA vaccine for HIV-1. We observed a dramatic increase in both antigen-specific T helper cell proliferation and CTL response. Eventual development of successful vaccines for HIV-1 would likely involve targeting multiple antigenic components of the virus to direct and empower the immune system to protect the host from viral infection. We present here the utility of multicomponent DNA immunization to elicit specific humoral and cell-mediated immune responses against different antigenic targets of HIV-1 as well as the ability of this immunization strategy to achieve significant enhancements of antigen-specific cellular immune responses.

Peptomers are polymers composed of peptides that are specifically cross-linked in a head-to-tail fashion. Recently, a peptomer composed of an amphipathic peptide from the C4 domain of HIV-1MN gp120 was shown to display a prominent alpha-helical conformation that, as an immunogen, elicited rabbit antibodies recognizing native and recombinant gp120 [Robey et al. (1995) J. Biol. Chem. 270, 23918-23921]. For the present study, we synthesized a conjugate composed of the C4 peptomer covalently linked to calcinated aluminum oxide nanoparticles. The nanoparticles were first reacted with (3-aminopropy])-triethoxysilane to provide an amine load of 15.9 mmol of R-NH2/g of solid. The amine-modified aluminum oxide nanoparticles then were reacted with N-acetylhomocysteine thiolactone at pH 10 to place a reactive thiol on the nanoparticles. A bromoacetylated C4 peptomer, modified at the epsilon-amines of lysine residues, then was reacted with the thiolated nanoparticles to give the peptomer covalently linked to aluminum oxide via a thioether bond. The peptomer load was determined to be 16 mg of peptomer/g of particles, a 55% theoretical yield. Particle shape and size of the peptomer-conjugated alumina were analyzed by electron microscopy and displayed a mean maximum diameter of 355 nm and a mean minimum diameter of 113 nm, well within the desired size range of 300 nm believed to be optimal for mucosal immunization purposes. Experimentally determined values of mean particle diameters, specific surface area, and specific peptomer load provided the information necessary to calculate the mean antigen load, which was determined to be 53000 +/- 42000 peptomer epitopes per particle. Peptomer-alumina conjugates, such as that described here, could form the basis of a new class of biomaterial that combines a chemically defined organic immunogen with a nontoxic chemically defined inorganic adjuvant.

Objectives: To evaluate the effect of sequence variation within the gp120 V3 loop on CD4 T-cell recognition., Design: CD4 T-cell clones were generated using synthetic peptides to circumvent the difficulties of using polyclonal T-cell responses. Peptides based on other HIV isolates were then used to determined the influence of single and multiple sequence differences., Results: Three of the panels of T-lymphocyte clones (TLC), which were all specific to diverse HIV-1 clade B gp120 V3-loop peptides differing in a limited number of residues, had heterogeneous patterns of response to peptides differing in length and sequence indicating that they recognized distinct but overlapping epitopes. The panels of TLC also differed in the extent to which they tolerated sequence differences between cell-culture-adapted or primary HIV-1 isolates. One panel responded to peptides based on several clade B and one clade D isolate. In contrast, two panels, generated from two different donors using the same peptide, only responded to a limited number of clade B isolates, whereas another only recognized HIV-1BRU. Two of the panels were also stimulated by peptides based on clinical isolates from one patient with some sequence changes enhancing T-cell recognition., Conclusions: These data are consistent with highly diverse CD4 T-cell recognition of the HIV-1 gp120 V3 loop, which is influenced by the sequence differences within the T-cell epitopic region and has implications for the pathogenesis and design of vaccines against HIV-1.