Faculty Bibliography

The V3 region of HIV-1 gp120 is important for virus-coreceptor interaction and highly immunogenic. Although most anti-V3 antibodies neutralize only the sensitive Tier 1 viruses, anti-V3 antibodies effective against the more resistant viruses exist, and a better understanding about these antibodies and their epitopes would be beneficial for the development of novel vaccine immunogens against HIV. The HIV-1 isolate JRFL with its cryptic V3 is resistant to most V3-specific monoclonal antibodies (mAbs). However, the V3 mAb 2424 achieves 100% neutralization against JRFL. 2424 is encoded by IgH V3-53 and IgL V2-28 genes, a pairing rarely used by the other V3 mAbs.. 2424 also has distinct binding and neutralization profiles. Studies of 2424-mediated neutralization of JRFL produced with a mannosidase inhibitor further revealed that its neutralizing activity is unaffected by the glycan composition of the virus envelope. To understand the distinct activity of 2424, we determined the crystal structure of 2424 Fab in complex with a JRFL V3 peptide, and showed that the 2424 epitope is located at the tip of the V3 crown (307IHIGPGRAFY318), dominated by interactions with HisP308, ProP313, and ArgP315. The binding mode of 2424 is similar to that of the well-characterized 447-52D, although 2424 is more side-chain dependent. The 2424 epitope is focused on the very apex of V3, away from nearby glycans, facilitating antibody access. This feature distinguishes the 2424 epitope from the other V3 crown epitopes and indicates that the tip of V3 is a potential site to target and incorporate into HIV vaccine immunogens. IMPORTANCE: HIV/AIDS vaccines are crucial for controlling the HIV epidemics that continue to afflict millions of people worldwide. However, HIV vaccine development has been hampered by significant scientific challenges, one of which is the inability of HIV vaccine candidates evaluated thus far to elicit production of potent and broadly neutralizing antibodies. The V3 loop is one of the few immunogenic targets on the virus envelope glycoprotein that can induce neutralizing antibodies, but in many viruses, parts of V3 are inaccessible for antibody recognition. This study examined a V3-specific monoclonal antibody that can completely neutralize HIV-1 JRFL, a virus isolate resistant to most V3 antibodies. Our data reveal that this antibody recognizes the most distal tip of V3 that is not as occluded as other parts of V3. Hence, the epitope of 2424 is in one of the vulnerable sites on the virus that may be exploited in designing HIV vaccine immunogens.

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.

Broadly neutralizing antibodies targeting the HIV-1 envelope (Env) are key components for protection against HIV-1. However, many cross-reactive epitopes are often occluded. This study investigates the mechanisms contributing to the masking of V2i (V2-integrin) epitopes compared to V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin alpha4beta7-binding motif on the V1V2 loop of HIV-1 Env gp120. The V2i monoclonal antibodies (mAbs) display extensive cross-reactivity with gp120 monomers from many subtypes, but neutralize only few viruses, indicating V2i's cryptic nature. First, we asked whether CD4-induced Env conformational changes affect V2i epitopes similar to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 mAbs, but not to the V2i mAbs. Second, contribution of N-glycans on masking V2i versus V3 epitopes was evaluated by testing neutralization of pseudoviruses produced in the presence of a glycosidase-inhibitor, kifunensine. Viruses grown in kifunensine were more sensitive to neutralization by V3 but not V2i mAbs. Finally, we evaluated the time-dependent dynamics of the V2i and V3 epitopes. Extending the time of virus-mAb interaction to 18 hours, before adding target cells, increased virus neutralization by some V2i mAbs and all V3 mAbs tested. Consistent with this, V2i mAb binding to Env on the surface of transfected cells also increased in time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic but distinct factors modulate antibody accessibility of these epitopes. The study reveals the importance of the structural dynamics of V2i and V3 epitopes in determining HIV-1 neutralization by antibodies targeting these sites. IMPORTANCE: Conserved neutralizing epitopes are present in the V1V2 and V3 regions of HIV-1 Env, but these epitopes are often occluded from Abs. This study reveals that distinct mechanisms contribute to the masking of V3 epitopes and V2i epitopes in the V1V2 domain. Importantly, V3 mAbs and some V2i mAbs display greater neutralization against relatively resistant HIV-1 isolates when the mAbs interact with the virus for a prolonged period of time. Given their highly immunogenic nature, V3 and V2i epitopes are valuable targets that would augment the efficacy of HIV vaccines.

Background: The RV144 clinical trial was shown to have an estimated vaccine efficacy of 31%. IgG antibodies (Abs) to V2 significantly inversely correlated with infection risk. Recently it was shown that Abs to V3 also inversely correlated with infection risk when vaccinees had low levels of Env-specific plasma IgA, and ADCC and neutralizing Abs. In addition, sieve analysis of breakthrough viruses identified significant differences in vaccine- and placebo-recipients in the proportion of certain residues present at positions 307 and 317 in V3. Studies were therefore performed to determine the fine specificity of V3 Abs in RV144 plasma and their possible role in reducing HIV infection. Methodology: Plasma specimens drawn two weeks after the last immunization from 40 vaccinees and 20 placebo recipients were tested in ELISA at a 1:100 dilution for reactivity vs. biotinylated cyclic V3 peptides (cV3s). Results: Of 8 cyclic V3 peptides studied, reactivity was strongest vs. clade B cV3s. The next strongest responses were with clade A, AG and C cV3s. The weakest responses were seen vs. clade AE cV3s (92TH023 & A244). The relative strengths of responses were consistent with analyses of the distance between the V3 sequences used in the assays. Plasma from placebo recipients were non-reactive. Seven variants of BaL and A244 cV3s with substitutions for I ³⁰⁷ and F ³¹⁷ were also tested. Vaccinees' plasma were most reactive with cV3 peptides containing I ³⁰⁷ (which was present in the vaccine), and much less reactive with substitutions (V, T or M) that were more frequent in vaccinees' breakthrough viruses than in those from placebo recipients. In contrast, plasma were equally reactive with wildtype A244 cV3 containing F ³¹⁷ and with A244 cV3 containing L ³¹⁷ despite the fact that vaccinee breakthrough viruses more frequently contained F ³¹⁷ than those from placebo recipients. Conclusions: RV144-induced V3 Abs were cross-reactive with cyclic V3 p!

The immune-correlate analysis of the RV144 clinical trial revealed that human plasma IgA immune responses elicited by the RV144 vaccine correlated positively with a risk for HIV acquisition. This result once again emphasized that HIV vaccines can potentially have adverse effects leading to enhancement of infection. Here, we discuss previously reported evidence of antibody-dependent enhancement of HIV infection. We also describe how a structure-based epitope-specific sieve-analysis can be employed to mine the molecular mechanism underlying this phenomenon.

HIV-1 envelope gp120 is the target for neutralizing antibodies (NAbs) against the virus. Various approaches have been explored to improve immunogenicity of broadly neutralizing epitopes on this antigen with limited success. We previously demonstrated that immunogenicity of gp120 and especially its V3 epitopes was enhanced when gp120 was co-administered as immune-complex vaccines with monoclonal antibodies (mAb) to the CD4-binding site (CD4bs). To define the mechanisms by which immune complexes influence V3 immunogenicity, we compared gp120 complexed with mAbs specific for the C2 region (1006-30), the V2 loop (2158), or the CD4bs (654), and found that the gp120/654 and gp120/2158 complexes elicited anti-V3 NAbs, but the gp120/654 complex was the most effective. gp120 complexed with 654 F(ab')2 was as potent, indicating that V3 immunogenicity is determined by the specificity of the mAb's Fab fragment used to form the complexes. Importantly, the gp120/654 complex not only induced anti-gp120 antibodies (Abs) to higher titers, but also of greater avidity. The Abs were cross-reactive with V3 peptides from most subtype B and some subtype C isolates. Neutralization was detected only against Tier-1 HIV-1 pseudoviruses, while Tier-2 viruses, including the homologous JRFL strain, were not neutralized. However, JRFL produced in the presence of a mannosidase inhibitor was sensitive to anti-V3 NAbs in the immune sera. These results demonstrate that the gp120/654 complex is a potent immunogen for eliciting cross-reactive functional NAbs against V3 epitopes, of which exposure is determined by the specific compositions of glycans shrouding the HIV-1 envelope glycoproteins.

Abstract Particulate matter (PM) varies in chemical composition and mass concentration based on a number of factors including location, season, source and particle size. The aim of this study was to evaluate the in vitro and in vivo toxicity of coarse and fine PM simultaneously collected at three rural and two urban sites within the metropolitan New York City (NYC) region during two seasons, and to assess how particle size and elemental composition affect toxicity. Human pulmonary microvascular endothelial (HPMEC-ST1.6R) and bronchial epithelial (BEAS-2B) cell lines were exposed to PM (50 mug/mL) and analyzed for reactive oxygen species (ROS). Mice (FVB/N) were exposed by oropharyngeal aspiration to 50 microg PM, and lavage fluid was analyzed for total protein and PMN influx. The ROS response was greater in the HPMEC-ST1.6R cell line compared to BEAS-2B cells, but the responses were significantly correlated (p < 0.01). The ROS response was affected by location, locale and the location:size interaction in both cell lines, and an additional association for size was observed from HPMEC-ST1.6R cells. Urban fine PM generated the highest ROS response. In the mouse model, inflammation was associated with particle size and by a season:size interaction, with coarse PM producing greater PMN inflammation. This study showed that the aerodynamic size, locale (i.e. urban versus rural), and site of PM samples affected the ROS response in pulmonary endothelial and epithelial cells and the inflammatory response in mice. Importantly, these responses were dependent upon the chemical composition of the PM samples.

Inhalation of ozone (O3), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O3 (2 parts/million). In wild-type mice, O3 exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O3-exposed wild-type mice. O3 exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O3 exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-beta-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O3 exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O3 and functionally contributes to the development of O3-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine.

One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.