Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes.

HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex and N88 at the trimer base. Virus neutralization was also affected, especially by antibodies against V1V2, V3, and gp41. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations, V3 crown, and gp41 epitopes. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 and other Env epitopes.

Role of Immunoglobulin M and A Antibodies in the Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and -RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin isotypes capable of blocking infection. METHODS: We studied spike- and RBD-specific immunoglobulin isotypes in convalescent and acute plasma/serum samples using a multiplex bead assay. We also determined virus neutralization activities in plasma and serum samples, and purified immunoglobulin fractions using a vesicular stomatitis pseudovirus assay. RESULTS: Spike- and RBD-specific immunoglobulin (Ig) M, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency. CONCLUSION: IgG, IgM, and IgA are critical components of convalescent plasma used for treatment of coronavirus disease 2019 (COVID-19).

Multimethod Longitudinal HIV Drug Resistance Analysis in Antiretroviral-Therapy-Naive Patients.

The global intensification of antiretroviral therapy (ART) can lead to increased rates of HIV drug resistance (HIVDR) mutations in treated and also in ART-naive patients. ART-naive HIV-1-infected patients from Cameroon were subjected to a multimethod HIVDR analysis using amplification-refractory mutation system (ARMS)-PCR, Sanger sequencing, and longitudinal next-generation sequencing (NGS) to determine their profiles for the mutations K103N, Y181C, K65R, M184V, and T215F/Y. We processed 66 ART-naive HIV-1-positive patients with highly diverse subtypes that underlined the predominance of CRF02_AG and the increasing rate of F2 and other recombinant forms in Cameroon. We compared three resistance testing methods for 5 major mutation sites. Using Sanger sequencing, the overall prevalence of HIVDR mutations was 7.6% (5/66) and included all studied mutations except K65R. Comparing ARMS-PCR with Sanger sequencing as a reference, we obtained a sensitivity of 100% (5/5) and a specificity of 95% (58/61), caused by three false-positive calls with ARMS-PCR. For 32/66 samples, we obtained NGS data and we observed two additional mismatches made up of minority variants (7% and 18%) that might not be clinically relevant. Longitudinal NGS analyses revealed changes in HIVDR mutations in all five positive subjects that could not be attributed to treatment. In one of these cases, superinfection led to the temporary masking of a resistant virus. HIVDR mutations can be sensitively detected by ARMS-PCR and sequencing methods with comparable performances. Longitudinal changes in HIVDR mutations have to be considered even in the absence of treatment.

Functional and Structural Characterization of Human V3-Specific Monoclonal Antibody 2424 with Neutralizing Activity against HIV-1 JRFL.

UNLABELLED: 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 of 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 IGHV3-53 and IGLV2-28 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 ((307)IHIGPGRAFYT(319)), dominated by interactions with His(P308), Pro(P313), and Arg(P315). The binding mode of 2424 is similar to that of the well-characterized MAb 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, which 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.

Effects of hookah smoking on indoor air quality in homes.

INTRODUCTION: Hookahs (water pipes) are rapidly increasing in popularity worldwide. Evidence suggests that although perceived as safer than cigarette smoke, hookah smoke may be as, or even more, dangerous as cigarette smoke. METHODS: Air samples from 33 homes-11 where only hookah-smoking occurred, 12 with only cigarettes and 10 with no smoking-were collected to analyse concentrations of particulate matter (PM2.5), black carbon, elemental and organic carbon and carbon monoxide (CO). Air quality was assessed in rooms where smoking occurred and in an adjacent room. RESULTS: Hookah and cigarette smoking impaired home air quality. The rooms in which hookahs were smoked showed the highest concentrations for all pollutants. CO was significantly greater in the rooms where hookahs were smoked than in the cigarette-smoking rooms and the non-smoking households (p<0.05). In addition, CO levels in the rooms adjacent to where hookah was smoked were 2.5-fold to 4-fold greater than those in the smoking and non-smoking rooms of the cigarette homes (p<0.05). PM2.5 levels were also elevated in hookah homes compared to cigarette and non-smoking homes, although not significantly different. CONCLUSIONS: This study, the first of its kind, demonstrates potentially hazardous levels of home air pollution in rooms where hookahs are being smoked as well as in adjacent rooms. These levels were greater than those in cigarette smoking homes, raising concerns about potential negative health effects on all individuals living in homes where hookahs are smoked.

Distinct mechanisms regulate exposure of neutralizing epitopes in the V2 and V3 loops of HIV-1 envelope.

UNLABELLED: 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 (variable loop V2 integrin) epitopes compared to the accessibility of V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin α4ß7-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 similarly to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 MAbs but not to the V2i MAbs. Second, the contribution of N-glycans in masking V2i versus V3 epitopes was evaluated by testing the 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 h 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 a time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic, but distinct factors modulate the 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.

Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine.

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-ß-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.

The effect of particle size, location and season on the toxicity of urban and rural particulate matter.

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 µg/mL) and analyzed for reactive oxygen species (ROS). Mice (FVB/N) were exposed by oropharyngeal aspiration to 50 µg 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.

Vaccination with immune complexes modulates the elicitation of functional antibodies against HIV-1.

Introduction: Neutralizing antibodies (Abs) are one of the immune components required to protect against viral infections. However, developing vaccines capable of eliciting neutralizing Abs effective against a broad array of HIV-1 isolates has been an arduous challenge. Objective: This study sought to test vaccines aimed to induce Abs against neutralizing epitopes at the V1V2 apex of HIV-1 envelope (Env). Methods: Four groups of rabbits received a DNA vaccine expressing the V1V2 domain of the CRF01_AE A244 strain on a trimeric 2J9C scaffold (V1V2-2J9C) along with a protein vaccine consisting of an uncleaved prefusion-optimized A244 Env trimer with V3 truncation (UFO-BG.ΔV3) or a V1V2-2J9C protein and their respective immune complexes (ICs). These IC vaccines were made using 2158, a V1V2-specific monoclonal Ab (mAb), which binds the V2i epitope in the underbelly region of V1V2 while allosterically promoting the binding of broadly neutralizing mAb PG9 to its V2 apex epitope in vitro. Results: Rabbit groups immunized with the DNA vaccine and uncomplexed or complexed UFO-BG.ΔV3 proteins (DNA/UFO-UC or IC) displayed similar profiles of Env- and V1V2-binding Abs but differed from the rabbits receiving the DNA vaccine and uncomplexed or complexed V1V2-2J9C proteins (DNA/V1V2-UC or IC), which generated more cross-reactive V1V2 Abs without detectable binding to gp120 or gp140 Env. Notably, the DNA/UFO-UC vaccine elicited neutralizing Abs against some heterologous tier 1 and tier 2 viruses from different clades, albeit at low titers and only in a fraction of animals, whereas the DNA/V1V2-UC or IC vaccines did not. In comparison with the DNA/UFO-UC group, the DNA/UFO-IC group showed a trend of higher neutralization against TH023.6 and a greater potency of V1V2-specific Ab-dependent cellular phagocytosis (ADCP) but failed to neutralize heterologous viruses. Conclusion: These data demonstrate the capacity of V1V2-2J9C-encoding DNA vaccine in combination with UFO-BG.ΔV3, but not V1V2-2J9C, protein vaccines, to elicit homologous and heterologous neutralizing activities in rabbits. The elicitation of neutralizing and ADCP activities was modulated by delivery of UFO-BG.ΔV3 complexed with V2i mAb 2158.

Cross-clade HIV-1 neutralizing antibodies induced with V3-scaffold protein immunogens following priming with gp120 DNA.

The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3(C)-CTB), or with double combinations of V3-CTB immunogens that included V3(C)-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity.

Evidence of dysregulation of dendritic cells in primary HIV infection.

Myeloid and plasmacytoid dendritic cells (DCs) are important mediators of both innate and adaptive immunity against pathogens such as HIV. During the course of HIV infection, blood DC numbers fall substantially. In the present study, we sought to determine how early in HIV infection the reduction occurs and whether the remaining DC subsets maintain functional capacity. We find that both myeloid DC and plasmacytoid DC levels decline very early during acute HIV infection. Despite the initial reduction in numbers, those DCs that remain in circulation retain their function and are able to stimulate allogeneic T-cell responses, and up-regulate maturation markers plus produce cytokines/chemokines in response to stimulation with TLR7/8 agonists. Notably, DCs from HIV-infected subjects produced significantly higher levels of cytokines/chemokines in response to stimulation with TLR7/8 agonists than DCs from uninfected controls. Further examination of gene expression profiles indicated in vivo activation, either directly or indirectly, of DCs during HIV infection. Taken together, our data demonstrate that despite the reduction in circulating DC numbers, those that remain in the blood display hyperfunctionality and implicates a possible role for DCs in promoting chronic immune activation.

Immune profiles to distinguish hospitalized versus ambulatory COVID-19 cases in older patients.

A fraction of patients with COVID-19 develops severe disease requiring hospitalization, while the majority, including high-risk individuals, experience mild symptoms. Severe disease has been associated with higher levels of antibodies and inflammatory cytokines but often among patients with diverse demographics and comorbidity status. This study evaluated hospitalized vs. ambulatory patients with COVID-19 with demographic risk factors for severe COVID-19: median age of 63, >80% male, and >85% black and/or Hispanic. Sera were collected four to 243 days after symptom onset and evaluated for binding and functional antibodies as well as 48 cytokines and chemokines. SARS-CoV-2-specific antibody levels and functions were similar in ambulatory and hospitalized patients. However, a strong correlation between anti-S2 antibody levels and the other antibody parameters, along with higher IL-27 levels, was observed in hospitalized but not ambulatory cases. These data indicate that antibodies against the relatively conserved S2 spike subunit and immunoregulatory cytokines such as IL-27 are potential immune determinants of COVID-19.

Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1.

Human anti-V3 monoclonal antibodies (mAbs) generated from HIV-1 infected individuals display diversity in the range of their cross-neutralization that may be related to their immunogenetic background. The study of the immunoglobulin (Ig) variable region gene usage of heavy chains have shown a preferential usage of the VH5-51 gene segment which was detected in 35% of 51 human anti-V3 mAbs. In contrast, human mAbs against other envelope regions of HIV-1 (anti-Env), including the CD4-binding domain, the CD4-induced epitope, and gp41 preferentially used the VH1-69 gene segment, and none of them used the VH5-51 gene. Furthermore, the usage of the VH4 family by anti-V3 mAbs was restricted to only one gene segment, VH4-59, while the VH3 gene family was used at a significantly lower frequency by all of the analyzed anti-HIV-1 mAbs. Multivariate analysis showed that usage of VH gene segments was significantly different between anti-V3 and anti-Env mAbs, and compared to antibodies from healthy subjects. In addition, the anti-V3 mAbs preferentially used the JH3 and D2-15 gene segments. The preferential usage of selected Ig gene segments and the characteristic pattern of Ig gene usage by anti-V3 mAbs can be related to the conserved structure of the V3 region.

Role of IgM and IgA Antibodies in the Neutralization of SARS-CoV-2.

BACKGROUND: SARS-CoV-2 has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and - RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin (Ig) isotypes capable of blocking infection. METHODS: We studied spike- and RBD-specific Ig isotypes in convalescent and acute plasma/sera using a multiplex bead assay. We also determined virus neutralization activities in plasma, sera, and purified Ig fractions using a VSV pseudovirus assay. RESULTS: Spike- and RBD-specific IgM, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency. CONCLUSION: IgG, IgM and IgA are critical components of convalescent plasma used for COVID-19 treatment.

Immune Correlates of Disease Progression in Linked HIV-1 Infection.

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1+ Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified post-hoc in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.

Airport-related air pollution and noise.

To provide quantitative evidence of the impact on people of a neighboring metropolitan airport, La Guardia Airport (LGA) in New York City, (1) airborne particulate matter (PM) was measured to determine whether concentration differences could be detected between homes that are upwind and downwind of the airport; (2) 24-hr noise measurements were made in 12 homes near the airport; and (3) the impact of noise was assessed by a Community Wellness and Health Promotion Survey. Particulate matter concentrations were higher during active airport operating hours than during nonoperating hours, and the percent increase varied inversely with distance from the airport. Hourly differences between paired upwind and downwind sites were not remarkable. Residents living near the airport were exposed to noise levels as much as four times greater than those experienced by residents in a quiet, comparison home. Impulse noise events were detected from both aircraft and vehicular traffic. More than 55% of the people living within the flight path were bothered by aircraft noise, and 63% by highway noise; these were significantly higher percentages than for residents in the nonflight area. The change in PM concentrations with distance during operating compared with nonoperating hours; traffic-related impulse noise events; and the elevated annoyance with highway noise, as well as aircraft noise among residents in the flight path area, show airport-related motor vehicle traffic to be a major contributor to the negative impact of airports on people in the surrounding communities.

Characterization of AHR2 and CYP1A expression in Atlantic sturgeon and shortnose sturgeon treated with coplanar PCBs and TCDD.

Atlantic sturgeon and shortnose sturgeon co-occur in many estuaries along the Atlantic Coast of North America. Both species are protected under the U.S. Endangered Species Act and internationally on the IUCN Red list and by CITES. Early life-stages of both sturgeons may be exposed to persistent aromatic hydrocarbon contaminants such as PCBs and PCDD/Fs which are at high levels in the sediments of impacted spawning rivers. Our objective was to compare the PCBs and TCDD sensitivities of both species with those of other fishes and to determine if environmental concentrations of these contaminants approach those that induce toxicity to their young life-stages under controlled laboratory conditions. Because our previous studies suggested that young life-stages of North American sturgeons are among the more sensitive of fishes to coplanar PCB and TCDD-induced toxicities, we were interested in identifying the molecular bases of this vulnerability. It is known that activation of the aryl hydrocarbon receptor 2 (AHR2) in fishes mediates most toxicities to these contaminants and transcriptional activation of xenobiotic metabolizing enzymes such as cytochrome P4501A (CYP1A). Previous studies demonstrated that structural and functional variations in AHRs are the bases for differing sensitivities of several vertebrate taxa to aromatic hydrocarbons. Therefore, in this study we characterized AHR2 and its expression in both sturgeons as an initial step in understanding the mechanistic bases of their sensitivities to these contaminants. We also used CYP1A expression as an endpoint to develop Toxicity Equivalency Factors (TEFs) for these sturgeons. We found that critical amino acid residues in the ligand binding domain of AHR2 in both sturgeons were identical to those of the aromatic hydrocarbon-sensitive white sturgeon, and differed from the less sensitive lake sturgeon. AHR2 expression was induced by TCDD (up to 6-fold) and by three of four tested coplanar PCB congeners (3-5-fold) in Atlantic sturgeon, but less so in shortnose sturgeon. We found that expression of AHR2 and CYP1A mRNA significantly covaried after exposure to TCDD and PCB77, PCB81, PCB126, but not PCB169 in both sturgeons. We also determined TEFs for the four coplanar PCBs in shortnose sturgeon based on comparison of CYP1A mRNA expression across all doses. Surprisingly, the TEFs for all four coplanar PCBs in shortnose sturgeon were much higher (6.4-162 times) than previously adopted for fishes by the WHO.

Functional Antibody Response Against V1V2 and V3 of HIV gp120 in the VAX003 and VAX004 Vaccine Trials.

Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 ß-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.

Differential induction of anti-V3 crown antibodies with cradle- and ladle-binding modes in response to HIV-1 envelope vaccination.

The V3 loop in the HIV envelope gp120 is one of the immunogenic sites targeted by Abs. The V3 crown in particular has conserved structural elements recognized by cross-reactive neutralizing Abs, indicating its potential contribution in protection against HIV. Crystallographic analyses of anti-V3 crown mAbs in complex with the V3 peptides have revealed that these mAbs recognize the conserved sites on the V3 crown via two distinct strategies: a cradle-binding mode (V3C) and a ladle-binding (V3L) mode. However, almost all of the anti-V3 crown mAbs studied in the past were isolated from chronically HIV-infected individuals. The extents to which the two types of anti-V3 crown Abs are generated by vaccination are unknown. This study analyzed the prevalence of V3C-type and V3L-type Ab responses in HIV-infected individuals and in HIV envelope-immunized humans and animals using peptide mimotopes that distinguish the two Ab types. The results show that both V3L-type and V3C-type Abs were generated by the vast majority of chronically HIV-infected humans, although the V3L-type were more prevalent. In contrast, only one of the two V3 Ab types was elicited in vaccinated humans or animal models, irrespective of HIV-1 envelope clades, envelope constructs (oligomeric or monomeric), and protocols (DNA plus protein or protein alone) used for vaccinations. The V3C-type Abs were produced by vaccinated humans, macaques, and rabbits, whereas the V3L-type Abs were made by mice. The V3C-type and V3L-type Abs generated by the vaccinations were able to mediate virus neutralization. These data indicate the restricted repertoires and the species-specific differences in the functional V3-specific Ab responses induced by the HIV envelope vaccines. The study implies the need for improving immunogen designs and vaccination strategies to broaden the diversity of Abs in order to target the different conserved epitopes in the V3 loop and, by extension, in the entire HIV envelope.

CCR5 expression and duration of high risk sexual activity among HIV-seronegative men who have sex with men.

OBJECTIVES: To test the hypothesis that in comparison with those with shorter risk duration, individuals with longer HIV risk duration would have reduced susceptibility to HIV-1 infection as measured by CCR5 expression, and to evaluate whether variation in CCR5 expression could be explained by known genetic polymorphisms. DESIGN AND METHODS: A cross-sectional study of HIV-1 exposed but uninfected men who have sex with men. The risk duration was estimated from self-reported years since first receptive anal intercourse. CCR5 expression on peripheral blood CD4+ monocytes and T cells was determined by flow cytometry. The CCR5-Delta32 mutation and polymorphisms in the CCR5 promoter and CCR2 as well as the copy number of CCL3L1 were analyzed by polymerase chain reaction. Plasma levels of MIP-1alpha (CCL3), MIP-1beta (CCL4) and RANTES (CCL5) were also measured. As risk duration varied with age, analyses were restricted to 67 individuals aged 30-49 years. RESULTS: Multiple linear regression analyses, adjusted for age and race, showed a significant negative association between HIV risk duration and CCR5 expression on monocytes (P = 0.01), and in a separate model, a similar negative association with CCR5 expression on T cells (P = 0.03). Low CCR5 expression was attributable mainly to CCR5-Delta32 heterozygosity and the CCR5-59029G allele. CONCLUSIONS: We confirmed a role for reduced CCR5 expression in HIV-1 resistance. CCR5-Delta32 heterozygosity and the CCR5-59029G allele were significant predictors of low CCR5 expression. Individuals with high CCR5 expression who resisted infection despite long HIV risk duration form an interesting group within which to search for additional mechanisms of resistance to HIV infection.