Principal Neutralizing Determinant Research Articles

Analysis of human adenovirus type 19 associated with epidemic keratoconjunctivitis and its reclassification as adenovirus type 64.

Human adenovirus species D type 19 (HAdV-D19) has been associated with epidemic keratoconjunctivitis (EKC), a highly inflammatory infection of the ocular surface. Confusion exists regarding the origins of HAdV-D19. The prototype virus (HAdV-D19p) does not cause EKC, while a virus identified later with the identical serologic determinant is a significant ocular pathogen. High throughput genome sequencing and bioinformatics analysis were performed on HAdV-D19p and three HAdV-D19 EKC strains, and compared to the previously sequenced clinical isolate, HAdV-D19 (C) and HAdV-D37. Corneas of C57BL/6J mice were injected with HAdV-D19p, HAdV-D19 (C), or virus-free buffer, and inflammation assessed by clinical examination, flow cytometry, and cytokine ELISA. Confocal microscopy and real-time PCR of infected corneal cell cultures were used to test viral entry. HAdV-D19 (C) and the other clinical EKC isolates showed nearly 100% sequence identity. EKC strains diverged from HAdV-D19p in the penton base, E3, and fiber transcription units. Simplot analysis showed recombination between EKC-associated HAdV-D19 with HAdV-D37, HAdV-D22, and HAdV-D19p, the latter contributing only the hexon gene, the principal serum neutralization determinant. HAdV-D19p induced stromal keratitis in the C57BL/6J mouse, but failed to infect productively human corneal epithelial cells. These data led to retyping of the clinical EKC isolates with a HAdV-D19 hexon gene as HAdV-D64. HAdV-D19 associated with EKC (HAdV-D64) originated from a recombination between HAdV-D19p, HAdV-D37, and HAdV-D22, and was mischaracterized because of a shared hexon gene. HAdV-D19p is not infectious for corneal epithelial cells, thus explaining the lack of any association with keratitis.

Porcine reproductive and respiratory syndrome virus diversity of Eastern Canada swine herds in a large sequence dataset reveals two hypervariable regions under positive selection

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to be genetically highly variable, but knowledge of sequence diversity from Eastern Canada and its degree of genetic plasticity in or near the principal neutralizing epitope (PNE) in association with evolutionary selective pressure is limited. The purposes of our study were to investigate the extent of strain diversity, the existing glycotypes and the amino acid sites under selective evolutionary pressure in its encoded protein, GP5, for a dataset of 1301 sequences (1998–2009). This was addressed by partitioning and clustering into subgenotypes a large number of open reading frame 5 sequences from the province of Quebec and analyzing the content of these subgenotypes. The overall pairwise diversity was 12% and was comparable to what has been reported around the world. The mean diversity for sequences within subgenotypes was around 7%. No marked variations in subgenotype emergence could be observed through time. Thirty-eight GP5 glycotype patterns were observed which included a newly identified site at position N57 which was already present in 1998. These patterns possessed one to six N-glycosylation sites in total and could be located in eight different positions. No obvious grouping of glycotypes could be established in relation to subgenotypes. Positions N44 and N51 were confirmed to be fixed N-glycosylation positions, whereas other positions where found to be shifting and located in or near hypervariable regions (HVRs) 1 and 2. Both HVRs were under selective evolutionary pressure in half of all subgenotypes including vaccine-like groups. Conversely, the PNE flanked by both HVRs was well conserved among most subgenotypes demonstrating potential molecular constraint in a probable viral binding region. The analysis of this dataset increased knowledge of evolutionary change inferred from genetic data, more specifically regarding the implications of both HVRs in PRRSV diversity.

To Probe the Conformational Adaptability of Conserved G-P-G-R Segment in the V3 Loop of HIV-1

To design an effective HIV vaccine it is important to study the relationship between sequence diversity and conformations of principal neutralizing determinant (PND) peptides, the hypervariable region of gp120. Two fragments 318-327 and 315-329 of gp120 are mapped as PND and can induce HIV-1 specific cytotoxic lymphocyte activity that could kill virus infected cells. Consequently, the design of more ordered and biologically relevant conformation of immunogenic region from gp120-V3 loop may aid in the design of more effective immunogens for HIV-1 vaccine development. In order to enlighten optimal structural preferences we have explored the conformational adaptability of two fragments from V3 loop of gp120 that contains G-P-G-R sequence encompassing mainly the crown region. Nuclear Magnetic Resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have been used to map out the conformation in diverse solvent systems such as water and hexafluroacetone (HFA). In HFA, the larger fragment, 315-329 shows some degree of inclination towards the formation of secondary structure. This indicates that length of the fragment is crucial in attaining secondary structure, which may be responsible for V3 loop’s hypervariable status. However, in water, both the fragments do not show any specific conformational preferences.

NMR evidence of charge‐dependent interaction between various PND V3 and CCR5 N‐terminal peptides

The third variable (V3) loop is an important region of glycoprotein 120 (gp120) for many biological processes, as it contains the highly conserved GPGR sequence and it represents the binding site for human immunodeficiency virus 1 (HIV-1) antibodies and for CCR5 and CXCR4 host cell coreceptors. The interaction of the principal neutralizing determinant (PND) V3 with the chemokine receptor CCR5 N-terminal region has been reported to be crucial for HIV-1 infection. The goal of this study is to characterize the solution structures of three HIV-1 gp120 V3 subtype B peptides and their interaction with a nonsulfated N-terminal CCR5 peptide. NMR titration experiments revealed that the CCR5Nt-PND V3 interaction is dependent on the number of the positively charged V3 residues, which is in agreement with the observation that increase in positive charge in the V3 sequence correlates with the augmentation of the interaction. As expected for free peptides in solution, the peptides representing the PND V3 region of gp120 exhibit conformational flexibility, but they also exhibit a large number of NOEs which allowed convergence to a dominant conformation. The PND V3 peptides retain the U-turn conformation observed in the crystal structures of gp120 complexes independently of CCR5 presence. The interaction of different regions of the CCR5Nt peptide is gradually increasing proportionally to the positive charge increase in the V3 peptides. The data demonstrate that the PND V3 and CCR5Nt peptide sequences have propensities for interaction even in the absence of sulfated tyrosines and that their binding and selectivity is determined by simple electrostatic attraction mechanisms.

Glycosylation of HIV-1 gp120 V3 Loop: Towards the Rational Design of a Synthetic Carbohydrate Vaccine

A wide variety of proteins can bind high-mannose oligosaccharides and are broadly neutralizing against HIV-1. However, success in eliciting broadly neutralizing antibodies against HIV-1 has been limited to date. The rational design of an HIV-1 vaccine is based on the information gained through the structural analysis of antibodies complexed with their epitopes. Of particular interest to this review are the binding of mannosides to human monoclonal antibody 2G12 recognizing Man(9)GlcNAc(2) from HIV-1 gp120. It is widely recognized that T-cell-independent antigens carbohydrates are poorly immunogenic, and fail to induce memory. To increase the immunogenicity, carbohydrate antigens have to be coupled to a highly immunogenic carrier. The design of peptide carbohydrate mimotopes (mimetics of carbohydrate antigens) is one approach that is currently explored to elicit neutralizing antibodies. This work is concerned with existing structural data on Man(9)GlcNAc(2) as the most promising epitope (or glycotope). Structural analysis of various torsion angles of Man(9)GlcNAc(2) is explored. The focus is made primarily on the third variable region (V3 loop) of gp120 due to its crucial relevance for coreceptor usage, as a principal neutralizing determinant (PND), and for its conserved glycosylation sites N295, N302 and N332. Valuable structural information from glycosylation effects is taken into account for the development of a V3 loop rational structure-based vaccine strategy using N295 and N302 as potential conformational epitope.

Towards Covalent Vaccination: IMPROVED POLYCLONAL HIV NEUTRALIZING ANTIBODY RESPONSE INDUCED BY AN ELECTROPHILIC gp120 V3 PEPTIDE ANALOG

Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain. Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain (MN) approximately 50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by gp120. Pretreatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines.

Characterization of the B Cell Epitopes Associated with a Truncated Form of Pseudomonas Exotoxin (PE38) Used to Make Immunotoxins for the Treatment of Cancer Patients

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

Stabilization of the Biologically Active Conformation of the Principal Neutralizing Determinant of HIV-1IIIBContaining acis-Proline Surrogate: 1H NMR and Molecular Modeling Study

The V3 loop is part of the gp120 glycoprotein, an extracellular protein located on the membrane of the human immunodeficiency virus (HIV-1). This loop is significantly important in many biological processes of the virus and contains the principal neutralizing determinant (PND). The PND is one of the most variable regions of the envelope, and this is probably related to the ability of the HIV virus to escape the immunologic defenses of the target host. Particular attention has been paid to the central part of the V3 loop which contains a highly conserved GPGR/GPGQ sequence and represents the binding site for antibodies. Many attempts have been made to design synthetic peptides as mimics of the V3 loop capable of eliciting immune response. However, this strategy suffers from the great conformational flexibility small peptides have in solution, which together with bioavailability represents the most important limitation to the usefulness of synthetic peptides as drugs and as synthetic immunogens. The use of conformationally constrained peptides can alleviate this problem. Early works using NMR studies have shown that a V3(IIIB) loop-derived peptide is conformationally heterogeneous when free in water. Upon complexation with 0.5beta, a monoclonal neutralizing antibody specific for the HIV-1(IIIB) strain, it adopts a beta-hairpin conformation with the central proline forming a type VIb beta-turn. In this study, we report the design and characterization of a conformationally restricted peptide with a sequence identical to that previously described, but with thiazolidine derivatives replacing the proline. The affinity of the 2,2-dimethylthiazolidine derivative for 0.5beta demonstrates that this moiety can successfully be used to mimic the proline in a cis conformation. This peptide not only displays a high propensity to adopt a beta-hairpin conformation but also retains the type VIb RGPG beta-turn similar to that found in the native complex. These compounds could help in elaborating more efficient immunogens for HIV-1 synthetic vaccine development.

Spying the neutralizing epitopes on E2 N-terminal by candidate epitope-vaccines against classical swine fever virus

Our previous study proved that the N-terminal (aa693–711) of glycoprotein E2 contained sequential neutralizing epitopes. In this study, four candidate epitope-vaccines (EVs) were separately prepared and evaluated. Among them, epitope-vaccine EV-BC1a (BC1a: aa693–699) induced high level of epitope-specific neutralizing antibodies and exhibited similar protective capability with that induced by Chinese vaccine strain (C-strain). These results confirmed CKEDYRY (aa693–699) as a principal sequential neutralizing epitope on E2 N-terminal. Moreover, these findings also indicate that epitope-vaccine is a potent candidate strategy for marker vaccine against classical swine fever virus (CSFV).

HIV-1 Glycopeptides as Immunogens

One challenge in HIV-1 vaccine design is to identify epitopes able to induce neutralizing antibodies. HIV-1 glycopeptides represent a partial structure of the envelope glycoproteins that contains both peptide and carbohydrate motifs. Several pieces of evidence suggest that HIV-1 glycopeptides may constitute new neutralizing epitopes: 1) certain HIV-1 glycopeptides are highly conserved and are well accessible; 2) selected N-glycans around the V3 domain have been identified as neutralizing epitope for the broadly neutralizing antibody 2G12; and 3) N-glycans can mask unwanted epitopes, redirect the immune focus, and induce conformational epitopes. To explore this new territory for immunogen design, we have focused on exploring the V3 domain glycopeptides that correspond to the so-called principal neutralizing determinant (PND) and the gp41 C-terminal glycopeptides that are involved in viral membrane fusion. We have developed a novel chemoenzymatic method for constructing large homogeneous HIV-1 glycopeptides that are hitherto unavailable. Preliminary studies indicated that glycosylation affects the global conformations and enhances the beta-turn and/or loop structure of the V3 domain in buffer. We also observed that the N-glycans can protect the V3 domain against protease (furin and pronase) digestion. In addition, we found that glycosylation on C34 has a profound effect on its ability to form the six-helix bundles with N36. The interesting glycosylation effects observed urge further immunization studies with the glycopeptide immunogens. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Candidate peptide-vaccine induced potent protection against CSFV and identified a principal sequential neutralizing determinant on E2

Previously, two candidate multi-peptide-vaccines (MPVs) consisted of five overlapping synthetic peptides covering the antigenic domain B/C (aa693–777) on envelope protein E2 were prepared in our lab. And they successfully induced peptide-specific neutralizing antibodies and provided pigs with complete protection from the lethal challenge of virulent classical swine fever virus (CSFV) strain Shimen. In this study, these five peptides were conjugated to bovine serum albumin (BSA), with which five groups of pigs ( n = 10) were inoculated, respectively. Among these candidate peptide-vaccines (PVs), PV-BC1 (BC1: aa693–716) exhibited the most potent protective activity, PV-BC3, PV-BC4 and PV-BC5 (BC3: aa723–745; BC4: aa741–760; BC5: aa757–777) had weaker effects, while no effect of PV-BC2 (BC2: aa712–727) had been detected. Moreover, the polyclonal antibodies induced by PV-BC1 and PV-BC4 were capable of neutralizing C-strain virus in vitro. Thus, a principal sequential neutralizing determinant (aa693–716) and a minor sequential neutralizing determinant (aa741–760) were proved to lie in the antigenic domain B/C, which can be recruited into developing more effective “marker vaccine” by epitope-vaccine strategy. Our study also indicates that scanning with a panel of sequential peptide-immunogens is an effective method to locate sequential neutralizing epitopes.

The neutralizing epitope ELDKWA on HIV-1 gp41: Genetic variability and antigenicity

Viral evasion through mutations of neutralizing epitopes is a major challenge to AIDS vaccine development and clinical therapy. In this study, we analyzed the genetic variability of principal neutralizing epitope ELDKWA among 5393 HIV isolates in the HIV sequence database ( http://www.hiv.lanl.gov). It has been found that six out of seven frequent variants were able to cause the invalidation of famous neutralizing MAb 2F5. Moreover, escape mutations K665S and K665E, often observed in C-subtype and O-group strains, respectively, were reported more frequently in recent years. Furthermore, we studied the antigenicity of these variants. Polyclonal antibodies induced by a candidate multi-epitope-vaccine were able to react with the recombinant gp41, respectively, carrying these variants, indicating that “epitope cocktail” strategy is necessary and helpful in vaccine design.

V3: HIV's Switch-Hitter

The third variable region, V3, of the gp120 surface envelope glycoprotein is an approximately 35-residue-long, frequently glycosylated, highly variable, disulfide-bonded structure that has a major influence on HIV-1 tropism. Thus the sequence of V3, directly or indirectly, can determine which coreceptor (CCR5 or CXCR4) is used to trigger the fusion potential of the Env complex, and hence which cells the virus can infect. V3 also influences HIV-1's sensitivity to, and ability to escape from, entry inhibitors that are being developed as antiviral drugs. For some strains, V3 is a prominent target for HIV-1 neutralizing antibodies (NAbs); indeed, for many years it was considered to be the "principal neutralization determinant" (PND). Some efforts to use V3 as a vaccine target continue to this day, despite disappointing progress over more than a decade. Recent findings on the structure, function, antigenicity, and immunogenicity of V3 cast new doubts on the value of this vaccine approach. Here, we review recent advances in the understanding of V3 as a determinant of viral tropism, and discuss how this new knowledge may inform the development of HIV-1 drugs and vaccines.

Dual Spatial Folds and Different Local Structures of the HIV-1 Immunogenic Crown in Various Virus Isolates

Local and global structural properties of the HIV-1 principal neutralizing epitope were studied in terms of NMR spectroscopy data reported in literature for the HIV-Haiti and HIV-RF isolates. To this effect, the NMR-based method comprising a probabilistic model of protein conformation in conjunction with the molecular mechanics and quantum chemical computations was used for determining the ensembles of conformers matching the NMR requirements and energy criteria. As a matter of record, the high resolution 3D structure models were constructed for the HIV-Haiti and HIV-RF immuno- genic crowns, and their geometric parameters were collated with the ones of conformers derived previously for describing the conformational features of immunogenic tip of gp120 from Thailand and MN HIV-1 strains. The HIV-1 neutralization site was demonstrated to constitute in water solution highly flexible system sensitive to its environment. This inference is completely valid for the geometric space of dihedral angles where statistically significant differences in local structures of simulated conformers have been found for all virus isolates of interest. In spite of this fact, the stretch analyzed was shown to manifest a certain conservatism in the space of atomic coordinates, building up in four HIV-1 isolates two spatial folds similar to those observed in crystal for the V3 loop peptides bound to different neutralizing Fabs. The results are discussed in the light of literature data on HIV-1 neutralizing epitope structure.

HIV mucosal vaccine: nasal immunization with rBCG-V3J1 induces a long-term V3J1 peptide-specific neutralizing immunity in Th1- and Th2-deficient condition

In the vaccine strategy against HIV, the bacillus Calmette–Guerin (BCG), a live attenuated strain of Mycobacterium bovis, is considered to be one of the potential vectors for mucosal delivery of antigens. We analyzed the induction of antigen-specific antibodies by nasal immunization with recombinant BCG vector-based vaccine (rBCG-V3J1) that secretes the V3 principal neutralizing epitope of HIV [Proc. Natl. Acad. Sci. U. S. A. 92 (1063) 1995]. C57BL/6 mice were nasally immunized with the rBCG-V3J1 (10 μg) four times by weekly intervals. After 1 week of the final immunization, V3J1-specific IgG was seen in the serum. However, antigen-specific secretary and serum IgA antibodies were not induced. High levels of antigen-specific serum IgG responses were maintained for 6 months following the nasal immunization. Antigen-specific IgG-producing cells were detected in mononuclear cells isolated from spleen, nasal cavity, and salivary gland of the nasally vaccinized mice. Furthermore, antigen-specific IgG antibodies induced by nasal immunization with rBCG-V3J1 possessed the ability to neutralize HIV in vitro . Collectively, nasal immunization together with rBCG-V3J1 is considered to be a powerful vaccination regimen for the induction of effective V3J1-specific immune responses. We are currently investigating antigen-specific CTL activity and cytokine production following nasal vaccination with rBCG-V3J1.

Local Structural Properties of the V3 Loop of Thailand HIV-1 Isolate

The model of locally accurate conformation for the HIV-Thailand principal neutralizing determinant (PND) located within the V3 loop of the virus envelope protein gp 120 was built in terms of NMR spectroscopy data. To this end, the NMR-based conformational analysis of synthetic molecule representing the peptide copy of the fragment under study was carried out using the published sequential d connectivity data and values of spin-spin coupling constants. As a result, (i) the local structure for the V3 loop from Thailand isolate was determined, (ii) the conformations of its irregular segments were analyzed, and the secondary structure elements identified, (iii) the ensemble of conformers matching the experimental and theoretical data was derived for the stretch forming the neutralizing epitope of the HIV-Thailand PND, (iv) to estimate the probability of realizing each of these conformers in solution, the results obtained were collated with the X-ray data for corresponding segments in synthetic molecules imitating the central region of the HIV-MN PND as well as for homologous segments 39–44 in Bence-Jonce REI protein (BJRP), 41–46 in immunoglobulin λ (Igλ), and 50–55 in β-chain of horse hemoglobin (HH), (v) to find the conserved structural motifs inside diverse HIV-1 isolates, the structure determined was compared with the one derived earlier for the HIV-MN PND from NMR spectroscopy data, (vi) on the basis of all data obtained, the 3D structure model describing the set of biologically relevant conformations, which may present different antigenic determinants to the immune system in various HIV-1 isolates, was proposed for the immunogenic crown of the V3 loop. The results obtained are discussed in conjunction with the data on the structure for the HIV-1 PND reported in literature.

Cross-clade neutralizing antibody production against human immunodeficiency virus type 1 clade E and B′ strains by recombinant Mycobacterium bovis BCG-based candidate vaccine

The recombinant Mycobacterium bovis BCG (rBCG) vector-based vaccine secreting the V3 principal neutralizing epitope of human immunodeficiency virus type 1 (HIV-1) Japanese strain was reported to induce both humoral and cellular immune responses effectively [Proc. Natl. Acad. Sci. USA. 92 (1995) 10693]. The antigen-secreting rBCG system was applied to the V3 epitope of clade E HIV-1 in this study. The V3 sequence of 19 amino acids (aa) and 15aa fused with mycobacterial α-antigen was not secreted while 12aa and 11aa sequences were successfully secreted from BCG cells. Serum IgG from guinea pig which was immunized with 12aa epitope-secreting recombinant BCG neutralized the WHO reference strain as well as primary field isolates of clade E virus. The serum IgG could also neutralize Thai B (clade B′) strains which possessed a conserved GPGQ motif in their V3 sequences. These data suggest that the rBCG construct secreting the 12aa epitope is implicated in the development of a prophylactic vaccine in Thailand in which both clade E and B′ viruses are prevalent.

Antibody convergence along a common idiotypic axis in immunodeficiency virus and hepatitis C virus infections.

The anti-idiotypic antibody 1F7 selectively binds antibodies against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) gag, pol, and env proteins. We tested anti-hepatitis C virus (HCV) antibodies to investigate selection of the 1F7 idiotype on antibodies against other chronic pathogens. Twelve of 15 HCV-seropositive individuals co-infected with HIV had detectable antibodies against recombinant HCV core, 4 against HCV NS4 protein, and 3 against HCV NS3 protein. All four HCV-seropositive, non-HIV-infected individuals had antibodies against HCV core and NS4, while 3 had antibodies against NS3. The 1F7 idiotype was frequently present on antibodies against each of the HCV antigens in the HIV co-infected and non-HIV-infected groups. Antibodies against HCV, including antibodies recognizing the putative principal neutralizing determinant of HCV E2 protein, displayed skewed kappa/lambda light chain usage consistent with clonal dominance. These observations extend the association between expression of the 1F7 idiotype and abnormal B cell clonal dominance in HIV and SIV infection to HCV infection and suggest that early establishment of an oligoclonal antibody response against HCV may freeze the B cell repertoire, impair adaptation to emergent HCV variants, and favor escape from neutralizing antibodies. We also demonstrated that expression of the 1F7 idiotype extends beyond antibodies against multiple antigens of AIDS-causing retroviruses to include antibodies against multiple antigens of an unrelated chronic hepatitis virus. Thus, distinct pathogens establishing chronic infection in the face of strong humoral immune responses select antibodies along a common idiotypic axis of the immune network.

HIV mucosal vaccine: nasal immunization with rBCG-V3J1 induces a long term V3J1 peptide-specific neutralizing immunity in Th1- and Th2-deficient conditions.

In the vaccine strategy against HIV, bacillus Calmette-Guérin (BCG), a live attenuated strain of Mycobacterium bovis, is considered to be one of potential vectors for mucosal delivery of vaccine Ag. We analyzed the induction of the Ag-specific Ab response by nasal immunization with recombinant BCG vector-based vaccine (rBCG-V3J1) that can secrete the V3 principal neutralizing epitope of HIV. Mice were nasally immunized with rBCG-V3J1 (10 microg) three times at weekly intervals. Four weeks after the initial immunization, high titers of V3J1-specific IgG Abs were seen in serum. These high levels of HIV-specific serum IgG responses were maintained for >12 mo following nasal immunization without any booster immunization. V3J1-specific IgG-producing cells were detected in mononuclear cells isolated from spleen, nasal cavity, and salivary gland of the nasally vaccinated mice. Nasal rBCG-V3J1 also induced high levels of prolonged HIV-specific serum IgG responses in Th1 (IFN-gamma(-/-))- or Th2 (IL-4(-/-))-immunodeficient mice. Further, IgG3 was highest among V3 peptide-specific IgG subclass Ab responses in these immunodeficient mice as well as in wild-type mice. In addition, this Ag-specific serum IgG Abs induced by nasal immunization with rBCG-V3J1 possessed the ability to neutralize clinical isolate of HIV in vitro. These results suggested that the nasal rBCG-V3J1 system might be used as a therapeutic vaccine in addition to a prophylaxis vaccine for the control of AIDS.

Structure of an anti-HIV monoclonal Fab antibody fragment specific to a gp120 C-4 region peptide.

Structure of an anti-HIV monoclonal Fab antibody fragment specific to a gp120 C-4 region peptide.