Neutralizing antibodies mechanism of neutralization and protective activity against HIV-1

The role of the humoral immune response in prevention against HIV-1 infection is still incompletely understood. However, neutralizing antibodies to certain epitopes on HIV-1 envelope glycoproteins inhibit HIV-1 infection in vitro and in vivo. Passive administration of these antibodies by themselvesor in combination completely protected hu-PBL-SCID mice or macaques from intravenous, vaginal, as well as maternal-fetal mucosal transmission. All these studies provide direct experimental evidence that neutralizing antibodies are potentenough to prevent HIV infection, and strongly suggest that neutralizing-antibody-based vaccines could provide effective protection against HIV-1, despite the potent action of CTLs. Some neutralizing epitopes have been defined in vitro and in vivo. Unfortunately, none of the neutralizing-antibody-based candidate vaccines has been demonstrated to induce enough protective activity. Weak antigenicity and immunogenicity of neutralizing epitopes on native or recombinant proteins and other factors made it difficult to induce neutralizing-epitope-specific antibody responses in vivo enough to prevent against primary isolates. Recent studies indicated that HIV-1 variations resulted in escape from neutralization or the CTL responses, which may be the principal challenge for HIV-1 prevention. Epitope vaccine as a new strategy activating both arms of the immune system, namely, using the “principal neutralizing epitopes” and the CTL epitopes in combination, should provide new hope for developing an effective vaccine to halt the HIV-1 epidemic.     

Protective humoral immune responses to the human immunodeficiency virus induced in immunized pigs: a possible source of therapeutic immunoglobulin preparations.

The human immunodeficiency virus (HIV-1) induces progressive and fatal disease in infected hosts. Initially the human immune response appears to control HIV infection. This hypothesis is supported by the long latency period observed during HIV infection prior to development of the active disease state. Similarly the observation of fetal protection from HIV infection in some pregnant women who have high titered neutralizing antibody responses to the virus underscores the importance of the humoral response to HIV in limiting infection. Therefore antibody replacement therapy is likely to provide substantial clinical benefits in this and other infected populations. However, currently there is no safe source for human antibodies with the desired protective qualities necessary for passive immune therapies. For a passive immune therapy to be valid it must protect against a diverse collection of viral isolates. Such a task is likely to require a complex mixture of human antibodies or human substitute antibodies which are available in large quantities and target conserved regions of the viral envelope, such that protection from diverse isolates are realized. Porcine products have been used extensively in many human therapeutic replacement regimens. Their use is primarily due to genetic similarity of the two species at the amino acid level which results in a high acceptance of grafted prosthetics and excellent tolerance of repeatedly administered biologicals. Accordingly we have examined the immunoglobulin responses to the Human Immunodeficiency Virus (HIV-1) of the Yorkshire mixed breed pig. Immunized animals developed significant humoral immunity as judged by ELISA, Western blot, radioimmunoprecipitation, flow microfluorimetry as well as in functional assays including neutralization and syncytia inhibition. The high neutralizing activity obtained and the immunological similarity between human and porcine immunoglobulin suggests that further investigation into the use of porcine immunoglobulin as human replacement antibodies for the therapeutic treatment of HIV is warranted.     

Analysis of the neutralizing antibody response elicited in rabbits by repeated inoculation with trimeric HIV-1 envelope glycoproteins

The elicitation of broadly neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, gp120 and gp41, remains a major challenge. Attempts to utilize monomeric gp120 as an immunogen to elicit high titers of neutralizing antibodies have been disappointing. Envelope glycoprotein constructs that better reflect the trimeric structure of the functional envelope spike have exhibited improved immunogenicity compared with monomeric gp120. We have described soluble gp140 ectodomain constructs with a heterologous trimerization motif; these have previously been shown to elicit antibodies in mice that were able to neutralize a number of HIV-1 isolates, among them primary isolate viruses. Recently, solid-phase proteoliposomes retaining the envelope glycoproteins as trimeric spikes in a physiologic membrane setting have been described. Here, we compare the immunogenic properties of these two trimeric envelope glycoprotein formulations and monomeric gp120 in rabbits. Both trimeric envelope glycoprotein preparations generated neutralizing antibodies more effectively than gp120. In contrast to monomeric gp120, the trimeric envelope glycoproteins elicited neutralizing antibodies with some breadth of neutralization. Furthermore, repeated boosting with the soluble trimeric formulations resulted in an increase in potency that allowed neutralization of a subset of neutralization-resistant HIV-1 primary isolates. We demonstrate that the neutralization is concentration-dependent, is mediated by serum IgG and that the major portion of the neutralizing activity is not directed against the gp120 V3 loop. Thus, mimics of the trimeric envelope glycoprotein spike described here elicit HIV-1-neutralizing antibodies that could contribute to a protective immune response and provide platforms for further modifications to improve the efficiency of this process.     

Humoral immune responses in human HIV-1 infection clearance of initial burst of virus replication and protection against disease progression.

An attempt is made to summarize the evidence that humoral immune responses in natural HIV-1 infection play a role in clearance of the initial burst of replication as well as in protection against rapid disease progression. Therefore, the so-called "asymptomatic carrier state" was defined on the basis of immunological characteristics, such as CD4+ cell number, CD45RA-CD29+ cell number, CD4+ proliferative responses to anti-CD3 mAbs and soluble activation markers, as well as virological characteristics such as the state of the viral genome in the cell, levels of genomic RNA production, antigenemia, viremia and virus phenotype. During natural infection two major classes HIV-1 neutralizing and cell-fusion inhibiting antibodies are elicited. One population directed against mostly continuous epitopes localized in the third variable domain (V3) of the envelope and one against discontinuous epitopes of the envelope. The last population blocks gp120-CD4 attachment, the first does not. The role of each of these populations of functional antibodies, in the clearance of viremia and the maintenance of the asymptomatic carrier state is discussed.     

Expansion of epitope cross-reactivity by anti-idiotype modulation of the primary humoral response

The primary humoral response produces antigen-specific antibodies so to clear the initial infection, and generates a population of corresponding memory cells to prevent infection by future encounters with the same pathogen. The continuous genetic modification of a pathogen's exterior, however, is one mechanism used to evade the immune defenses of its host. Here we describe a novel means, involving anti-idiotypic antibodies, by which the host can counteract such pathogen genetic alterations by modulation of its primary humoral response. An autoimmune response against primary antibodies, Ab1's, creates anti-idiotypic antibodies (Ab2's), some of which (designated Ab2alpha) are able to bind the Ab1/antigen complex. We have discovered that binding of Ab2alpha to its corresponding Ab1 can expand Ab1's ability to bind variations of its antigen. This expanded epitope cross-reactivity is shown not only to increase the binding activity of Ab1 but also its ability to neutralize a variant infectious virus. MAb M77 is an Ab1, which is highly strain-specific for the HIV-1 envelope protein gp120(IIIB). This Ab1 can be rendered cross-reactive and neutralizing for an otherwise resistant HIV strain by its interaction with a unique anti-idiotypic Ab2alpha (GV12). Furthermore, molecular characterization of this expanded cross-reactivity was accomplished using combinatorial phage display peptide libraries.     

Structural insights into key sites of vulnerability on HIV-1 Env and influenza HA

Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor-binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals.     

Epitope-vaccine strategy against HIV-1: today and tomorrow

Vaccines play important roles in preventing infectious diseases caused by different pathogens. However, some pathogens such as HIV-1 challenge current vaccine strategy. Poor immunogenicity and the high mutation rate of HIV-1 make great difficulties in inducing potent immune responses strong enough to prevent infection via vaccination. Epitope-vaccine, which could intensively enhance predefined epitope-specific immune responses, was suggested as a new strategy against HIV-1 and HIV-1 mutation. Epitope-vaccines afford powerful approaches to elicit potent, broad and complete immune protection against not only primary homologous viral isolates but also heterologous viral mutants. Although most studies are still preliminary now, epitope-vaccine as a novel strategy against the AIDS epidemic has great developmental potential. To trigger T-cell-dependent IgG antibody responses and improve affinities of the epitope-specific antibodies, approaches such as recombinant multi-epitope-vaccination and prime-boosting vaccination were suggested. Cellular immune responses, especially CTL responses, could also be elicited and enhanced in addition to humoral immune responses. Developed epitope-vaccines activating both arms of the immune system would benefit prevention and immunotherapy not only against HIV but also other chronic infections.     

Comparison of immunoblots with neutralizing and complement fixing antibodies in experimental and natural cases of visna-maedi

Summary In this study the humoral antibody response in visna-maedi virus disease in sheep during long-term infection was analyzed utilizing immunoblot assays, neutralization tests and complement fixation tests. In immunoblot assays antibodies to several virus specific protein bands were detected, both against the viral envelope glycoproteins and internal proteins of the virus. The immunoblot reaction pattern resembled that found in HIV-1 infection in humans, consistent with reported similar molecular weight of the major proteins of these two viruses. The immunoblot band pattern was compared with the pattern of complement fixing and neutralizing antibodies through the preclinical and clinical course in natural and experimental cases of visna-maedi. Of six immunoblot bands identified as virus specific, the antibody response against threegag products and the majorenv glycoprotein appeared early in infection, at a similar time as the complement fixing antibodies. The response against two proteins, one presumably the transmembrane protein and the other possibly agag precursor, was delayed.     

Structure-based,targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization.     

Immunogenicity of recombinant envelope glycoproteins derived from T-cell line-adapted isolates or primary HIV isolates: a comparative study using multivalent vaccine approaches

We investigated immunogenic properties of native envelope glycoproteins derived from HIV-1 (subtype B). Our main objective was to assess whether the design of multivalent vaccines affects generation of neutralizing antibodies against primary viruses. Recombinant Semliki Forest virus (SFV) particles producing various HIV-1 envelope glycoproteins were used as vaccine vectors. The following multivalent vaccination approaches were compared: 1) immunization with a mixture of recombinant SFV expressing envelope glycoproteins derived from three HIV-1 primary isolates and two T-cell laboratory-adapted (TCLA) viruses; 2) immunization with a mixture of recombinant SFV expressing only the envelope glycoproteins derived from three HIV-1 primary isolates; 3) sequential immunizations with the recombinant SFV expressing the envelope glycoproteins derived from three HIV-1 primary isolates and two TCLA viruses, respectively. Two monovalent vaccine approaches using SFV expressing envelope glycoproteins derived from a single primary isolate or TCLA virus were also included in the study. The multivalent vaccination strategies based on SFV vaccine vectors did not induce more neutralizing antibodies than the previously tested TCLA envelope immunogens, which gave disappointing results against primary isolates.     

ELNKWA-epitope specific antibodies induced by epitope-vaccine recognize ELDKWA- and other two neutralizing-resistant mutated epitopes on HIV-1 gp41

Based on the fact that monoclonal antibody (mAb) 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 separately or in combination with other mAbs showed potent neutralizing activity to a wide range of primary HIV-1 isolates in vivo and in vitro, but this epitope undergoes restricted mutation. ELNKWA is a neutralizing-resistant mutated epitope. We induced ELNKWA-epitope-specific polyclonal and monoclonal antibodies and studied the interaction of the antibodies with ELDKWA-epitope and other two neutralizing-resistant mutated epitopes. The candidate ELNKWA-epitope-vaccine induced a high level of antibodies to the ELNKWA-epitope-peptide. The ELNKWA-epitope-specific polyclonal antibodies bound not only the ELNKWA-, but also ELDKWA-, ELEKWA- and ELDEWA-epitope-peptides in ELISA-assay. Moreover, the antibodies also recognized four C-domain-peptides (P5, P6, P7, P8) which contain these four epitopes, respectively. Interestingly, an ELNKWA-epitope-specific monoclonal antibody (TH-Ab1) induced by the candidate ELNKWA-epitope-vaccine could also recognize the four C-domain-peptides containing ELNKWA-, ELDKWA-, ELEKWA- and ELDEWK-epitopes. These results indicate that the candidate ELNKWA-epitope-vaccine could induce high levels of antibodies, which recognize the neutralizing epitope ELDKWA and three neutralizing-resistant mutated epitopes, suggesting that the candidate ELNKWA-epitope-vaccine may help to overcome the problem of viral escape from neutralization through mutation at D or K position, and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1.     

Candidate Antibody-Based Therapeutics Against HIV-1

Antibody-based therapeutics have been successfully used for the treatment of various diseases and as research tools. Several well characterized, broadly neutralizing monoclonal antibodies (bnmAbs) targeting HIV-1 envelope glycoproteins or related host cell surface proteins show sterilizing protection of animals, but they are not effective when used for therapy of an established infection in humans. Recently, a number of novel bnmAbs, engineered antibody domains (eAds), and multifunctional fusion proteins have been reported which exhibit exceptionally potent and broad neutralizing activity against a wide range of HIV-1 isolates from diverse genetic subtypes. eAds could be more effective in vivo than conventional full-size antibodies generated by the human immune system. Because of their small size (12～15?kD), they can better access sterically restricted epitopes and penetrate densely packed tissue where HIV-1 replicates than the larger full-size antibodies. HIV-1 possesses a number of mechanisms to escape neutralization by full-size antibodies but could be less likely to develop resistance to eAds. Here, we review the in vitro and in vivo antiviral efficacies of existing HIV-1 bnmAbs, summarize the development of eAds and multispecific fusion proteins as novel types of HIV-1 inhibitors, and discuss possible strategies to generate more potent antibody-based candidate therapeutics against HIV-1, including some that could be used to eradicate the virus.     

Role of Humoral Immunity in Host Defense Against HIV

Individuals infected with HIV-1 and nearly everyone vaccinated with HIV-1 vaccines will, in time, generate antibodies against viral proteins. These antibodies do not resolve natural infection, and vaccine candidates that successfully stimulate the production of high titers of neutralizing antibodies have failed to protect against infection. In spite of this, antibodies continue to be a focus of vaccine research. One reason for the continued interest in antibodies is the failure of a vaccine engineered to generate cell-mediated immunity against HIV. Successful protective immunity against most intracellular pathogens involves several arms of the immune response. A successful vaccine should also stimulate both protective cell-mediated immunity and specific antibody. Efforts should be directed toward making a vaccine that will stimulate the production of 1) more antibody, 2) more broadly cross-reactive neutralizing antibody (broadly neutralizing antibodies), and 3) antibody with a particular functional activity (antibody-dependent cell-mediated cytotoxicity; catalytic antibodies).     

Induction of V3-Specific Cytotoxic T Lymphocyte Responses by HIVgagParticles Carrying Multiple Immunodominant V3 Epitopes of gp120

Efforts to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimericgag–envvirus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2gagprecursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1_IIIB, HIV-1_MN, HIV-1_RF, and HIV-1_SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purifiedgag–envVLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than thegagVLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1_IIIB, HIV-1_MN, HIV-1_RF, and the gp120 antiserum against HIV-1_SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2gagcan be used as a potential HIV vaccine against various HIVs.     

Selective recognition of oligomeric HIV-1 primary isolate envelope glycoproteins by potently neutralizing ligands requires efficient precursor cleavage

A critical component of an effective HIV vaccine will be the induction of broadly neutralizing antibodies. Comprising the HIV spike, the exterior envelope glycoprotein gp120 and the transmembrane glycoprotein gp41 mediate receptor binding, viral entry, and are the targets for neutralizing antibodies. The gp120 and gp41 glycoproteins are derived from the gp160 precursor glycoprotein and following gp160 glycosylation, oligomerization and cleavage in the endoplasmic reticulum and Golgi, remain as non-covalently associated trimers of heterodimers. Previously, using cell-surface envelope glycoproteins derived from infection of a laboratory-adapted HIV-1 strain, a correlation had been established between the binding of gp120-directed antibodies to the viral glycoprotein and the ability of the antibodies to neutralize laboratory-adapted isolates. However, this has been more difficult to demonstrate for glycoproteins derived from primary patient isolates. Here, using a FACS-based method, we report that only gp120-directed neutralizing antibodies and the neutralizing ligand soluble CD4 efficiently bind to glycoproteins derived from the JR-FL primary isolate provided that the gp160 precursor protein is efficiently cleaved. Precursor cleavage was demonstrated by cell-surface biotinylation and Western blotting. In stark contrast, both non-neutralizing and neutralizing antibodies bind non-cleaved envelope glycoproteins from JR-FL and YU2 isolates. These data imply that significant changes in Env spike structure are dependent upon precursor gp160 cleavage and are consistent with a restricted-binding-to-Env model of neutralization. The data also have implications in regards to the use and design of non-cleaved envelope glycoprotein trimeric immunogens as a means to selectively and preferentially present neutralizing epitopes to the host immune system.     

IMMUNOGENICITY AND SPECIFICITY OF THE CANDIDATE MULTI-EPITOPE-VACCINES AGAINST HIV-1

The failure of some candidate HIV-1 vaccines may result from inducing very weak neutralization activity against representative primary viral isolates. Based on our hypothesis that epitope-vaccine may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, we designed two candidate multi-epitope-vaccines, EP1 [C-G-(ELDKWA-GPGRAFY)₂-K] and EP2 (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD), containing three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope protein, and expected them to induce epitope-specific antibodies of predefined epitope-specificity. The two peptides were conjugated to carrier protein bovine serum albumin (BSA) and used for immunization of rabbits. Proteins were purified from the rabbit sera induced by both candidate multi-epitope-vaccines (EP1-BSA and EP2-BSA) through affinity chromatography with epitope-peptide-conjugated sepharose-column, and identified as antibodies in silver-staining and immunoblotting. These antibodies were demonstrated to recognize three neutralizing epitopes on peptides and the recombinant gp41 in ELISA-assay and immunoblotting. These results indicated that both candidate multi-epitope-vaccines could induce high levels of antibodies of predefined epitope-specificity which recognized a few of neutralizing epitopes on peptides and protein, providing experimental evidence for the new strategy to develop an effective neutralizing-antibody-based multi-epitope-vaccine against HIV-1.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1).

Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-1) are considered important for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-1 gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-1 are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-1. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-1 isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-1 IIIB. The results suggest the possibility of developing broadly protective HIV-1 immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.     

Site-directed primary in vitro immunization: production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors.

The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120 (gp120), linked to a promiscuous helper T-cell epitope of tetanus toxin. The peptide was used to induce a human humoral in vitro immune response against the V3 region, using lymphocytes obtained from healthy, sero-negative blood donors. The in vitro immunized peripheral blood lymphocytes were Epstein-Barr virus infected and the antibody response to the synthetic peptide was evaluated using a solid-phase ELISA with the recombinant C-terminal fragment of gp120 (pB1, amino acid residues 287-467, derived from the HIV-1 LAI isolate). The heterotope construct yielded a significantly frequency of specifically immunized B cells, in contrast to the control immunizations with individual T and B epitopes, mixtures of these epitopes or no immunogen at all. This approach allowed us to generate human monoclonal antibodies, using lymphocytes derived from sero-negative donors, that cross-neutralized several HIV-1 strains, inhibited syncytia formation as well as prevented spreading of the viral infection from cell to cell. Thus, site-directed in vitro immunization using synthetic heterotopes might prove valuable in the dissection and induction of a protective humoral immune response.     

Human immunodeficiency virus-1 specific and natural cellular immunity in HIV seronegative subjects with multiple sexual exposures to virus.

The probability of HIV infection by sexual contact, although it varies greatly, appears to be lower than that of infection by other routes of exposure. The aim of this study was to evaluate immunological determinants involved in protection against HIV infection in subjects with multiple and repeated sexual exposures to the virus. Twenty-two subjects were studied for CD8+ cell anti-HIV suppression activity and serum neutralizing activity against the HIV strain of their own partners, beta-chemokine production, and natural killer cell activity. CD8+ cell anti-HIV activity and neutralizing activity of sera were found in 13 (76%) and 12 (70.5%) out of 17 HIV-1 negative subjects, respectively. Six individuals had a relevant immune response against HIV: three subjects with a high CD8+ cell antiviral suppression activity and three individuals with sera neutralizing activity titer >1:10. These last three subjects had the highest beta-chemokine levels, a very prolonged period of multiple sexual intercourse (>6 years) and a seropositive partner with a high viral load. A partial reduction of neutralizing activity titer was observed when pre-incubating the sera with anti-beta-chemokine neutralizing antibodies. A spontaneous natural killer cell activity was suppressed in the majority of HIV-1 negative subjects with sexual exposure in comparison with normal individuals. The protection from sexual HIV transmission appears to be the result of a network of different humoral and cellular factors.     

Polyvalent HIV-1 Env vaccine formulations delivered by the DNA priming plus protein boosting approach are effective in generating neutralizing antibodies against primary human immunodeficiency virus type 1 isolates from subtypes A, B, C, D and E

A major challenge in developing an HIV-1 vaccine is to identify immunogens and their delivery methods that can elicit broad neutralizing antibodies against primary isolates of different genetic subtypes. Recently, we demonstrated that priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. In the current study, we examined whether the DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing one, three or eight primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. Our results suggest that DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1 vaccines to elicit neutralizing antibody responses against viruses with diverse genetic sequence variations.