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CCR5 is the main co-receptor for HIV-1 cell entry and it plays key roles in HIV-1 mucosal transmission. Natural anti-CCR5 antibodies were found in HIV-1-exposed seronegative and long-term non-progressor subjects, suggesting a role in controlling viral replication in vivo. We assessed the effect of sera containing or not natural anti-CCR5 antibodies, on membrane CCR5 level and HIV-1 infection in primary macrophages. Sera modulated CCR5 expression with a trend dependent on the donor/serum tested but independent on the presence or absence of anti-CCR5 antibodies. All sera strongly reduced HIV-1 DNA in all donor's macrophages and no correlation was observed between CCR5 and viral DNA levels. These results suggest that CCR5 expression level is not a major determinant of macrophage infection and that the observed modulation of CCR5 and HIV-1 DNA might depend on factors other than CCR5-reactive antibodies present in sera and/or intrinsic to the donors on which sera were tested., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Recent studies showed that immunological tolerance may restrict the development of Env-specific autoreactive broadly neutralizing antibodies. This evidence is consistent with the finding that Env immunization of a systemic lupus erythematosus (SLE) murine model produced antibodies that neutralize tier 2 HIV-1 strains. In this study, we address the possibility of eliciting neutralizing anti-Env antibodies in other autoimmune diseases such as multiple sclerosis (MS). While, as reported for SLE, we showed for the first time that a small number of HIV-1 negative, relapsing remitting MS patients exhibited antibodies with neutralizing properties, our attempts at inducing those antibodies in a EAE mouse model of MS failed. The success in eliciting Env-specific neutralizing antibodies might be related to the specific characteristics of the autoimmune disease, or it might rely in improving the vaccination design. Studies using mouse models are useful to gain insight in how HIV-specific neutralizing antibody responses are regulated in order to develop a protective HIV-1 vaccine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Siracusano, Finardi, Pastori, Martinelli, Furlan and Lopalco.)

Objectives: Few studies have investigated chronically infected individuals after antiretroviral therapy (ART) interruption (ATI, analytical therapy interruption); thus, we investigated the association between some HIV-specific antibodies and viral control., Design: All enrolled patients were previously described in the APACHE study. Briefly, the study was conducted on HIV-1 chronically infected patients, with HIV-RNA less than 50 copies/ml for at least 10 years, CD4+ cell count greater than 500 cells/μl and HIV-DNA less than 100 copies/106 PBMC. The ART regimen in use at the time of ATI was resumed at confirmed viral rebound (CVR, defined as two consecutive HIV-RNA >50 copies/ml)., Methods: Collection of sera and analysis of both binding antibodies (BAbs) and neutralizing antibodies (NAbs) was performed at three different time points: ATI, CVR and time of viral re-suppression after ART resumption., Results: IgG subclasses (IgG1, IgG2, IgG3 and IgG4) from the four patients with highest levels of neutralization were found to block viral infection. All patients had CVR after ATI at a median time of 21 days (14-56). After ART resumption, all the enrolled patients achieved HIV-RNA less than 50 copies/ml in 42 days (21-98). We observed a strong increase of either BAbs and NAbs titers from ATI to viral re-suppression in one patient, who showed the longest period of virus undetectability during ATI. In this patient, BAbs and NAbs specifically belonged to both IgG1 and IgG4 subclasses, directed to env antigen., Conclusion: env-specific NAbs and BAbs belonging to IgG1, IgG4 subclasses could be helpful to monitor long-term responses able to control virus replication and eradicate HIV infection., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Microbicides are considered a promising strategy for preventing human immunodeficiency virus (HIV-1) transmission and disease. In this report, we first analyzed the antiviral activity of the miniCD4 M48U1 peptide formulated in hydroxyethylcellulose (HEC) hydrogel in activated peripheral blood mononuclear cells (PBMCs) infected with R5- and X4-tropic HIV-1 strains. The results demonstrate that M48U1 prevented infection by several HIV-1 strains including laboratory strains, and HIV-1 subtype B and C strains isolated from the activated PBMCs of patients. M48U1 also inhibited infection by two HIV-1 transmitted/founder infectious molecular clones (pREJO.c/2864 and pTHRO.c/2626). In addition, M48U1 was administered in association with tenofovir, and these two antiretroviral drugs synergistically inhibited HIV-1 infection. In the next series of experiments, we tested M48U1 alone or in combination with tenofovir in HEC hydrogel with an organ-like structure mimicking human cervicovaginal tissue. We demonstrated a strong antiviral effect in absence of significant tissue toxicity. Together, these results indicate that co-treatment with M48U1 plus tenofovir is an effective antiviral strategy that may be used as a new topical microbicide to prevent HIV-1 transmission., Competing Interests: The authors declare no competing financial interests.

Objective: The frequency of immature transitional B cells is increased in blood of HIV-1-infected individuals. We investigated whether HIV-1 infection affects expression and function of chemokine receptors important for egress of immature transitional B cells from bone marrow and migration to lymphoid organs., Design: This is a cross-sectional study analysing the migratory phenotype and function of immature transitional B cells in HIV-1-infected individuals, in relation to antiretroviral treatment and age., Methods: Frequency of blood immature transitional B cells and their phenotypic characteristics, including chemokine receptors and a maturation marker, were determined by immunostainings. Migratory capacities were studied in a migration assay., Results: The increased frequency of immature transitional B cells in untreated HIV-1 infection was normalized in patients receiving antiretroviral treatment; in our cohorts, age did not have an impact on the frequency of circulating immature transitional B cells. Immature transitional B cells from nontreated patients expressed low levels of CD21 molecule. We found an elevated frequency of CXCR3 and CXCR4 expressing immature transitional B cells in treated and nontreated patients. CXCR4 receptor was unresponsive to CXCL12 ligand in in-vitro migration and internalization assays. In addition, CXCR5 expression was downregulated on immature transitional B cells from infected patients, and these cells migrated poorly in response to CXCR5 ligand., Conclusion: Circulating immature transitional B cells from HIV-1-infected patients are not fully mature, probably due to premature egress from bone marrow; these cells showed a phenotype which could impair entry into secondary lymphoid organs. Changes in migratory capacity of immature transitional B cells may affect B-cell maturation during HIV-1 infection.

Glycosphingolipids (GSLs) are involved in HIV-1 entry. GM-3 ganglioside, a widespread GSL, affects HIV entry and infection in different ways, depending on the concentration, through its anchoring activity in lipid rafts. This explains why the induction of an altered GSLs metabolism was a tempting approach to reducing HIV-1 cell infection. This study assayed the biological properties of a synthetic GM-3 lactone mimetic, 1, aimed at blocking HIV-1 infection without inducing the adverse events expected by an altered metabolism of GLSs in vivo. The mimetic, conjugated to immunogenic protein ovalbumin and multivalently presented, was able to bind the CD4 molecule with high affinity and block its engagement with gp120, thus inhibiting virus entry. Elicited antimimetic antibodies were also able to block HIV-1 infection in vitro, with activity complementary to that observed for 1. These preliminary results show that the use of GSLs mimetics can be a novel promising mode to block HIV-1 infection and that 1 and other GSL mimetics deserve further attention.

Background: Early events in HIV infection are still poorly understood; virus derived from acute infections, the transmitted/founders IMCs, could provide more reliable information as they represent strains that established HIV infection in vivo, and therefore are investigated to elucidate potentially shared biological features., Methods: This study examined synergy in neutralization by six monoclonal antibodies targeting different domains in gp120 and gp41 and assayed in pairwise combination against 11 HIV-1 clade B strains, either Env pseudoviruses (PV, n = 5) or transmitted/founder infectious molecular clones (T/F IMCs, n = 6). Three of the early-infection env tested as PV were juxtaposed with T/F viruses derived from the same three patients, respectively., Results: All antibodies reaching IC50 were assayed pairwise (n = 50). T/F IMCs showed overall lower sensitivity to neutralization by single antibodies than PV, including within the three patient-matched pairs. Remarkably, combination index (CI) calculated using the Chow and Talalay method indicated synergy (CI < 0.9) in 42 data sets, and occurred in T/F IMC at similar proportions (15 of 17 antibody-T/F IMC combinations tested) as in pseudoviruses (27 of 33). CI values indicative of additivity and low-level antagonism were seen in 5 and 3 cases, respectively. Most pairs showed comparable synergic neutralizing effects on both virus groups, with the 4E10 + PG16 pair achieving the best synergic effects. Variability in neutralization was mostly observed on pseudovirus isolates, suggesting that factors other than virus isolation technology, such as env conformation, epitope accessibility and antibody concentration, are likely to affect polyclonal neutralization., Conclusions: The findings from this study suggest that inhibitory activity of bNAbs can be further augmented through appropriate combination, even against viruses representing circulating strains, which are likely to exhibit a less sensitive Tier 2 neutralization phenotype. This notion has important implications for the design and development of anti-Env bNAb-inducing vaccines and polyclonal sera for passive immunization.

Introduction: Understanding the mechanisms by which some individuals are able to naturally control HIV-1 infection is an important goal of AIDS research. We here describe the case of an HIV-1(+) woman, CASE1, who has spontaneously controlled her viremia for the last 14 of her 20 years of infection., Methods: CASE1 has been clinically monitored since 1993. Detailed immunological, virological and histological analyses were performed on samples obtained between 2009 and 2011., Results: As for other Elite Controllers, CASE1 is characterized by low to undetectable levels of plasma HIV-1 RNA, peripheral blood mononuclear cell (PBMC) associated HIV-1 DNA and reduced in vitro susceptibility of target cells to HIV-1 infection. Furthermore, a slow rate of virus evolution was demonstrated in spite the lack of assumption of any antiretroviral agent. CASE1 failed to transmit HIV-1 to either her sexual male partner or to her child born by vaginal delivery. Normal values and ratios of T and B cells were observed, along with normal histology of the intestinal mucosa. Attempts to isolate HIV-1 from her PBMC and gut-derived cells were unsuccessful, despite expression of normal cell surface levels of CD4, CCRC5 and CXCR4. CASE1 did not produce detectable anti-HIV neutralizing antibodies in her serum or genital mucosal fluid although she displayed potent T cell responses against HIV-1 Gag and Nef. CASE1 also possessed multiple genetic polymorphisms, including HLA alleles (B*14, B*57, C*06 and C*08.02) and HLA-C single nucleotide polymorphisms (SNPs, rs9264942 C/C and rs67384697 del/del), that have been previously individually associated with spontaneous control of plasma viremia, maintenance of high CD4(+) T cell counts and delayed disease progression., Conclusions: CASE1 has controlled her HIV-1 viremia below the limit of detection in the absence of antiretroviral therapy for more than 14 years and has not shown any sign of immunologic deterioration or disease progression. Co-expression of multiple protective HLA alleles, HLA-C SNPs and strong T cell responses against HIV-1 proteins are the most likely explanation of this very benign case of spontaneous control of HIV-1 disease progression.

Background: Aged individuals respond poorly to vaccination and have a higher risk of contracting infections in comparison to younger individuals; whether age impacts on the composition and function of B cell subpopulations relevant for immune responses is still controversial. It is also not known whether increased age during HIV-1 infection further synergizes with the virus to alter B cell subpopulations. In view of the increased number of HIV-1 infected patients living to high age as a result of anti-retroviral treatment this is an important issue to clarify., Results: In this work we evaluated the distribution of B cell subpopulations in young and aged healthy individuals and HIV-1 infected patients, treated and naïve to treatment. B cell populations were characterized for the expression of inhibitory molecules (PD-1 and FcRL4) and activation markers (CD25 and CD69); the capacity of B cells to respond to activation signals through up-regulation of IL-6 expression was also evaluated. Increased frequencies of activated and tissue-like memory B cells occurring during HIV-1 infection are corrected by prolonged ART therapy. Our findings also reveal that, in spite of prolonged treatment, resting memory B cells in both young and aged HIV-1 infected patients are reduced in number, and all memory B cell subsets show a low level of expression of the activation marker CD25., Conclusions: The results of our study show that resting memory B cells in ART-treated young and aged HIV-1 infected patients are reduced in number and memory B cell subsets exhibit low expression of the activation marker CD25. Aging per se in the HIV-1 infected population does not worsen impairments initiated by HIV-1 in the memory B cell populations of young individuals.

Upon its mucosal entry, human immunodeficiency virus type 1 (HIV-1) is internalized by Langerhans cells (LCs) in stratified epithelia and transferred locally to T cells. In such epithelia, LCs are in direct contact with peripheral neurons secreting calcitonin gene-related peptide (CGRP). Although CGRP has immunomodulatory effects on LC functions, its potential influence on the interactions between LCs and HIV-1 is unknown. We show that CGRP acts via its receptor expressed by LCs and interferes with multiple steps of LC-mediated HIV-1 transmission. CGRP increases langerin expression, decreases selected integrins, and activates NF-κB, resulting in decreased HIV-1 intracellular content, limited formation of LC-T cell conjugates, and elevated secretion of the CCR5-binding chemokine CCL3/MIP-1α. These mechanisms cooperate to efficiently inhibit HIV-1 transfer from LCs to T cells and T cell infection. In vivo, HIV-1 infection decreases CGRP plasma levels in both vaginally SHIV-challenged macaques and HIV-1-infected individuals. CGRP plasma levels return to baseline after highly active antiretroviral therapy. Our results reveal a novel path by which a peripheral neuropeptide acts at the molecular and cellular levels to limit mucosal HIV-1 transmission and suggest that CGRP receptor agonists might be used therapeutically against HIV-1.

Unlabelled: Mucosal antibodies harboring various antiviral activities may best protect mucosal surfaces against early HIV-1 entry at mucosal sites and they should be ideally induced by prophylactic HIV-1 vaccines for optimal prevention of sexually transmitted HIV-1. A phase I, double-blind, randomized, placebo-controlled trial was conducted in twenty-four healthy HIV-uninfected young women. The study objectives were to assess the safety, tolerability and immunogenicity of virosomes harboring surface HIV-1 gp41-derived P1 lipidated peptides (MYM-V101). Participants received placebo or MYM-V101 vaccine at 10 μg/dose or 50 μg/dose intramuscularly at week 0 and 8, and intranasally at week 16 and 24. MYM-V101 was safe and well-tolerated at both doses administered by the intramuscular and intranasal routes, with the majority of subjects remaining free of local and general symptoms. P1-specific serum IgGs and IgAs were induced in all high dose recipients after the first injection. After the last vaccination, vaginal and rectal P1-specific IgGs could be detected in all high dose recipients. Approximately 63% and 43% of the low and high dose recipients were respectively tested positive for vaginal P1-IgAs, while 29% of the subjects from the high dose group tested positive for rectal IgAs. Serum samples had total specific IgG and IgA antibody concentrations ≥ 0.4 μg/mL, while mucosal samples were usually below 0.01 μg/mL. Vaginal secretions from MYM-V101 vaccinated subjects were inhibiting HIV-1 transcytosis but had no detectable neutralizing activity. P1-specific Th1 responses could not be detected on PBMC. This study demonstrates the excellent safety and tolerability of MYM-V101, eliciting systemic and mucosal antibodies in the majority of subjects. Vaccine-induced mucosal anti-gp41 antibodies toward conserved gp41 motifs were harboring HIV-1 transcytosis inhibition activity and may contribute to reduce sexually-transmitted HIV-1., Trial Registration: ClinicalTrials.gov NCT01084343.

Natural antibodies to gp41 inhibit HIV-1 replication through the recognition of two different regions, corresponding to the leucine zipper motif in the HR1 alpha-helix and to another motif within HR2 region, hosting 2F5 and 4E10 epitope. This study aimed at reproducing such protective responses through VLP vaccination. Six regions covering the alpha-helical regions of gp41 were conjugated to the surface of AP205 phage-based VLPs. Once administered in mice via systemic or mucosal route, these immunogens elicited high titers of gp41-specific IgG. Immunogenicity and HIV infectivity reduction were obtained either with HR2 regions or with peptides where aminoacid strings were added to either the C-terminus or N-terminus of core epitope in HR1 region. Antibody-dependent cell cytotoxicity (ADCC) activity was induced by one of the HR2 epitopes only. These results may have relevant implications for the development of new vaccinal approaches against HIV infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The constant heavy chain (CH1) domain affects antibody affinity and fine specificity, challenging the paradigm that only variable regions contribute to antigen binding. To investigate the role of the CH1 domain, we constructed IgA2 from the broadly neutralizing anti-HIV-1 2F5 IgG1, and compared 2F5 IgA2 and IgG binding affinity and functional activities. We found that 2F5 IgA2 bound to the gp41 membrane proximal external region with higher affinity than IgG1. Functionally, compared with IgG1, 2F5 IgA2 more efficiently blocked HIV-1 transcytosis across epithelial cells and CD4(+) cell infection by R5 HIV-1. The 2F5 IgG1 and IgA2 acted synergistically to fully block HIV-1 transfer from Langerhans to autologous CD4(+) T cells and to inhibit CD4(+) T-cell infection. Epitope mapping performed by screening a random peptide library and in silico docking modeling suggested that along with the 2F5 IgG canonical ELDKWA epitope on gp41, the IgG1 recognized an additional 3D-conformational epitope on the gp41 C-helix. In contrast, the IgA2 epitope included a unique conformational motif on the gp41 N-helix. Overall, the CH1 region of 2F5 contributes to shape its epitope specificity, antibody affinity, and functional activities. In the context of sexually transmitted infections such as HIV-1/AIDS, raising a mucosal IgA-based vaccine response should complement an IgG-based vaccine response in blocking HIV-1 transmission.

MicroRNAs (miRNAs) inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4(+) T cells from HIV-1 élite long-term nonprogressors (éLTNPs), naive patients, and multiply exposed uninfected (MEU) patients, and we observed that the éLTNP patients clustered with naive patients, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU patients and 23 miRNAs distinguished naive from MEU patients, whereas only 1 miRNA (miR-155) discriminated éLTNP from naive patients. We proposed that miRNA expression may discriminate between HIV-1-infected and -exposed but negative patients. Analysis of miRNAs expression after exposure of healthy CD4(+) T cells to gp120 in vitro confirmed our hypothesis that a miRNA profile could be the result not only of a productive infection but also of the exposure to HIV-1 products that leave a signature in immune cells. The comparison of normalized Dicer and Drosha expression in ex vivo and in vitro condition revealed that these enzymes did not affect the change of miRNA profiles, supporting the existence of a Dicer-independent biogenesis pathway.

HIV-exposed, uninfected (EUN) babies born to HIV-infected mothers are examples of natural resistance to HIV infection. In this study, we evaluated the titer and neutralizing potential of gp41-specific maternal antibodies and their correlation with HIV transmission in HIV-infected mother-child pairs. Specific gp41-binding and -neutralizing antibodies were determined in a cohort of 74 first-time mother-child pairs, of whom 40 mothers were infected with HIV subtype C. Within the infected mother cohort, 16 babies were born infected and 24 were PCR negative and uninfected at birth (i.e., exposed but uninfected). Thirty-four HIV-uninfected and HIV-unexposed mother-child pairs were included as controls. All HIV-positive mothers and their newborns showed high IgG titers to linear epitopes within the HR1 region and to the membrane-proximal (MPER) domain of gp41; most sera also recognized the disulfide loop immunodominant epitope (IDE). Antibody titers to the gp41 epitopes were significantly lower in nontransmitting mothers (P < 0.01) and in the EUN babies (P < 0.005) than in HIV-positive mother-child pairs. Three domains of gp41, HR1, IDE, and MPER, elicited antibodies that were effectively transmitted to EUN babies. Moreover, in EUN babies, epitopes overlapping the 2F5 epitope (ELDKWAS), but not the 4E10 epitope, were neutralization targets in two out of four viruses tested. Our findings highlight important epitopes in gp41 that appear to be associated with exposure without infection and would be important to consider for vaccine design.

Elicitation of a potent and broadly neutralizing antibody response is the main goal of an effective preventive HIV-1 vaccine. It has been shown by us and others that the expression of Env glycoproteins on the surface of particulate structures, such as Virus-Like Particles (VLPs), could be a more efficient strategy to deliver conformational epitopes to the immune system. To this aim, VLPs expressing native HIV Env gp140 or gp41 glycoproteins have been produced in insect cells using a baculovirus expression system and characterized for appropriate protein expression. VLP-bound HIV gp140 glycoprotein showed the appropriate expression and trimeric conformation. Immunogenicity studies have been performed in BALB/C mice by intra-peritoneal administration and sera from immunized mice have been tested in ELISA assays, for their reactivity with HIV specific antigens, as well as in ex vivo neutralization assay. Sera from immunized animals showed a high reactivity with individual HIV proteins expressed in VLPs. Results of TZM-bl based neutralization assay show that combined sera from animals independently immunized with gp140- or full-length-gp41-expressing VLPs have an additive/synergistic effect in the neutralization activity of HIV pseudoviruses. In conclusion, novel VLPs expressing different HIV Env glycoproteins with native trimeric conformation have been generated, showing the induction of effective antibody response with neutralization activity in TZM-bl neutralization assay. These results confirm the effectiveness of VLPs as presentation and delivery system for conformational proteins and show the improved neutralization activity upon the combination of anti-sera elicited by different HIV envelope antigens, suggesting the possibility of broadening the spectrum of viral epitopes targeted by immune response., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To address an existing controversy concerning the presence of HIV-1-specific antibodies of the IgA isotype in the female genital tract secretions of highly-exposed but persistently seronegative (HEPSN) women, 41 samples of plasma and cervicovaginal lavage (CVL) fluid were distributed to six laboratories for their blinded evaluation using ELISA with 10 different HIV-1 antigens, chemiluminescence-enhanced Western blots (ECL-WB), and virus neutralization. HIV-specific IgG or IgA antibodies in plasma samples from HEPSN women were absent or detectable only at low levels. In CVL, 11/41 samples displayed low levels of reactivity in ELISA against certain antigens. However, only one sample was positive in two of five laboratories. All but one CVL sample yielded negative results when analyzed by ECL-WB. Viral neutralizing activity was either absent or inconsistently detected in plasma and CVL. Plasma and CVL samples from 26 HIV-1-infected women were used as positive controls. Irrespective of the assays and antigens used, the results generated in all laboratories displayed remarkable concordance in the detection of HIV-1-specific antibodies of the IgG isotype. In contrast, IgA antibodies to HIV-1 antigens were not detected with consistency, and where present, IgA antibodies were at markedly lower levels than IgG. Although HIV-neutralizing activity was detected in plasma of all HIV-1-infected women, only a few of their CVL samples displayed such activity. In conclusion, frequent HIV-1 sexual exposure does not stimulate uniformly detectable mucosal or systemic HIV-1-specific responses, as convincingly documented in the present blindly performed study using a broad variety of immunological assays. Although HIV-1-infection leads to vigorous IgG responses in plasma and CVL, it does not stimulate sustained IgA responses in either fluid.

Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.

Objectives: Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines produced during inflammatory conditions, may impact on IL-7 production., Design: We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST., Results: We found that IFN-gamma enhanced the expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant down-regulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL-1alpha,IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs and of important chemo-attractants for T cells. The microarray results were verified by additional methods., Conclusions: Our results are discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.

Objectives: To determine whether oral HIV-1 exposure incites a persistent systemic anti-HIV-1 response in exposed uninfected individuals of discordant couples of men who have sex with men, and whether this response associates with HIV-1 exposure measured by viral load in the HIV-positive partners., Methods: Plasma were collected from exposed uninfected individuals (n = 25), HIV-positive partners (n = 25) and low-risk controls (n = 22). A peripheral blood mononuclear cells-based neutralization assay was used to test these samples against three primary HIV-1 isolates. Self-reported questionnaires described routes of HIV-1-exposure, and clinical records documented viral loads in HIV-positive partners., Results: At enrollment, plasma samples from seven of 25 exposed uninfected individuals neutralized at least two of the three HIV-1 isolates. No samples from the 22 controls neutralized any HIV-1 isolate (P = 0.01). Of these seven exposed uninfected individuals, six retained neutralization capacity during follow-up. Neutralization capacity among exposed uninfected individuals associated with the highest measured viral load of their respective partners (P = 0.01) and also time since highest viral load (P = 0.02). Purified plasma immunoglobulin (Ig) A1-mediated neutralization was observed in six of the seven samples, whereas none of the IgA1-depleted plasma samples neutralized HIV-1. The neutralizing IgA1 was not HIV envelope specific as detected by ELISA and western blot., Conclusion: Orally exposed uninfected men who have sex with men can mount neutralizing anti HIV-1 activity in plasma, mediated primarily by non-HIV envelope-specific IgA1. Neutralization was associated with previous measured highest viral load in the HIV-positive partner, as well as time elapsed since the peak viral load. Neutralization also persisted over time in spite of a continuous low viral exposure.

The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors. Infectability by HIV (strains IIIB and Bal and primary isolates) was significantly reduced (P=0.016) in silenced cells compared with cells that maintained HLA-C expression in 10 of the 11 PBL donors. Normal infectability was resumed, together with HLA-C expression, when the effect of siRNA interference waned after several days in culture. Additional confirmation of the HLA-C effect was obtained in several assays employing HLA-C-positive and -negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with env genes from HIV strains AC10 and QH0692.42 were assayed on siRNA-silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses.

HIV-1 exposure causes several dramatic unbalances in the immune system homeostasis. Here, we will focus on the paradox whereby CD4 specific autoimmune responses, which are expected to contribute to the catastrophic loss of most part of the T helper lymphocyte subset in infected patients, may display the characteristics of an unconventional protective immunity in individuals naturally resistant to HIV-1 infection. Reference to differences in fine epitope mapping of these two oppositely polarized outcomes will be presented, with particular reference to partially or totally CD4-gp120 complex-specific antibodies. The fine tuning of the anti-self immune response to the HIV-1 receptor may determine whether viral exposure will result in infection or, alternatively, protective immunity.Along this line, an efficacious anti-HIV strategy can rely on the active (i.e., through immunization) or passive targeting of cryptic epitopes of the CD4-gp120 complex, including those harboured within the CD4 molecule. Such epitopes are expected to be safe from genetic drift and thus allow for broad spectrum of efficacy. Moreover, since these epitopes are not routinely exposed in uninfected individuals, they are expected to become targets of neutralizing antibodies or other specifically designed molecules only after viral exposure, with a predictable low impact in terms of potentially harmful anti-CD4 self-reactivity.The experimentum naturae of naturally resistant individuals indicates a strategy to design innovative strategies to neutralize HIV-1 by acting on the sharp edge between harmful and protective self-reactivity.

AIDS is mainly a sexually transmitted disease, and accordingly, mucosal tissues are the primary sites of natural human immunodeficiency virus type-1 (HIV-1) transmission. Mucosal immunoglobulin A (IgA) antibody specific for HIV-1 envelope gp41 subunit is one correlate of protection in individuals who are highly sexually exposed to HIV-1 but remain persistently IgG seronegative (HEPS). Understanding these peculiar IgAs at the gene and functional level is possible only with monoclonal IgAs. We have constructed a mucosal Fab IgA library from HEPS and have characterized a series of HIV-1 IgAs specific for gp41 that, in vitro, are transcytosis-blocking and infection-neutralizing. Characterization of their IgA genes shows that Fab specific for the gp41 membrane-proximal region harbors a long heavy-chain CDR3 loop (CDRH3) similar to the two broadly neutralizing IgG monoclonal antibodies, 2F5 and 4E10. Furthermore, the selected Fab IgA shows extensive somatic mutations that cluster in the CDR regions, indicating that affinity maturation due to an antigen-driven process had occurred in HEPS individuals, presumably upon multiple exposures to HIV. This analysis of HEPS monoclonal IgA gives a unique opportunity to correlate an antibody function (resistance to a pathogen in vivo) with an antibody gene. Such neutralizing monoclonal IgAs could be used in microbicide formulation.

Envelope-based immunogens capable of generating high titers of neutralizing antibodies have until now been difficult to generate, or failed to act as useful vaccines to prevent HIV-1 infection and disease progression. On the other hand, humoral immune responses to self and allogeneic cellular antigens involved in HIV-1 docking and entry are present both in infected patients and in subjects with natural resistance to HIV-1 infection, where they share similarities but also display definite differences. By dissecting these subtle differences, crucial cellular and molecular markers, possibly correlated with natural resistance to HIV-1 and with the modulation of clinical progression in stably infected patients, have been identified. Here, state-of-the art knowledge on anti-self immune responses following infection or exposure to HIV will be reviewed. The possible implications of these mechanisms in the design of unconventional therapies aimed to counteract the peculiar HIV-1 capability to circumvent the immune system will be discussed.

Human natural killer (NK) (CD3- CD56+) cells can be divided into two functionally distinct subsets, CD3- CD56(dim) and CD3- CD56(bright). We analysed the distribution of NK cell subsets in primary and chronic human immunodeficiency virus-1 (HIV-1) infection, to determine if HIV infection stage may influence the subset distribution. In primary infection, contrary to chronic infection, the CD3- CD56(dim) subset was expanded compared to healthy controls. We also studied the effect of antiretroviral therapy administered early in infection and found that NK cell subset distribution was partially restored after 6 months of antiretroviral therapy in primary infection, but not normalized. Recently, NK cells have been divided into CD27- and CD27+ subsets with different migratory and functional capacity and CD27-mediated NK cell activation has been described in mice. We therefore investigated whether CD27 and/or CD70 (CD27 ligand) expression on NK cells, and thus the distribution of these novel NK subsets, was altered in HIV-1-infected patients. We found up-regulated expression of both CD27 and CD70 on NK cells of patients, resulting in higher proportions of CD27(high) and CD70(high) NK cells, and this phenomenon was more pronounced in chronic infection. Experiments conducted in vitro suggest that the high interleukin-7 levels found during HIV-1 infection may participate in up-regulation of CD70 on NK cell subsets. Imbalance of NK cell subsets and up-regulated expression of CD27 and CD70 initiated early in HIV-1 infection may indicate NK cell activation and intrinsic defects initiated by HIV-1 to disarm the innate immune response to the virus.

Serum IL-7 levels correlate with T-cell depletion in HIV-infected individuals. In some patients, we observed that serum IL-7 decreases upon progression to AIDS, suggesting a role for IL-7 in T-cell maintenance in sporadic cases. Interestingly, IL-7 levels were significantly lower in stable long-term non-progressors (LTNP) than in patients who lost the LTNP status in a 3-year follow-up (P < 0.001), indicating that the serum IL-7 concentration might be a valuable marker for maintenance of the LTNP state.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a human cytidine deaminase, is a potent inhibitor of HIV replication. To explore a possible role of this protein in modulating in vivo susceptibility to HIV infection, we analyzed APOBEC3G expression in HIV-exposed seronegative individuals, HIV-seropositive patients, and healthy control subjects. The results showed that the expression of APOBEC3G is significantly increased in peripheral blood mononuclear cells (PBMCs)--mainly CD14(+) cells--and in cervical tissues of HIV-exposed seronegative individuals. Higher APOBEC3G expression correlated with a reduced susceptibility of PBMCs to in vitro infection with the HIV-1(Ba-L) R5 strain. APOBEC3G could be important in modulating in vivo susceptibility to sexually transmitted HIV infection.

Objective: The genital mucosa represents the major site for initial host-HIV-1 contact. HIV-1-protective mucosal immunity has been identified either in subjects who despite repeated sexual exposure, remain seronegative (ESN) or in long-term non-progressor HIV-1-seropositive individuals (LTNP). As a subset of ESN and LTNP produce anti-CCR5 antibodies both at systemic and mucosal level, we studied the role of anti-CCR5 antibodies in blocking HIV transfer through human epithelial cells., Design and Methods: To evaluate HIV-1-inhibitory activity by anti-CCR5 antibodies, a two-chambers system was established to model HIV-1 infection across the human mucosal epithelium. Moreover, peripheral blood mononuclear cells (PBMC) and a CCR5 transfected cell line were also used in a classical HIV-infectivity assay. CCR5-specific IgG and IgA were used to inhibit HIV replication., Results: Either serum or mucosal IgA to CCR5 were able to specifically block transcytosis of CCR5- but not CXCR4-HIV strains across a tight epithelial cell layer by interacting with the first extracellular loop of the receptor (amino acids YAAAQWDFGNTMCQ). Monoclonal antibodies against other regions of CCR5 had no effect on HIV transcytosis. Moreover, mucosal CCR5-specific IgA neutralized CCR5-tropic strains and SOS-JRFL pseudovirus replication in PBMC and CCR5 transfected cell lines respectively, with a mechanism different than that observed for transcytosis., Conclusions: Anti-CCR5 Abs shed light on the immunological mechanisms involved in the control of HIV-1 infection in a model that can be considered an experimentum naturae for resistance to HIV. They could be useful in the design of new strategies against HIV infection at mucosal sites.

Objective: The expression of innate immune molecules associated with potential blocking activity of HIV-1 propagation was analyzed in the cervical tissue of a group of African HIV-1 IgG-negative commercial sex workers (CSWs) with an HIV-1-encountering risk behavior., Methods: Cervical biopsies from the superior portion of the ectocervix were assessed for innate immune molecules and evaluated in situ by computerized image analysis at the single-cell level., Results: A higher expression of interferon-alpha (IFNalpha) and RANTES was detected in CSWs and HIV-1-infected individuals as compared to low-risk HIV-1-uninfected controls (Neg Ctrls). Most (>90%) of RANTES-expressing cells were CD8 cells as determined by confocal microscopy. In contrast, the expression of leukemia inhibitory factor (LIF) and secretory leukocyte protease inhibitor (SLPI) was comparable between the groups. The expression of beta-defensin 2 was highest in HIV-1-infected individuals., Conclusions: Induction of IFNalpha and RANTES expression in cervical mucosa may contribute to protection of sexual HIV-1 transmission in subjects with a higher risk behavior.

Objectives: To investigate the effects of primary HIV-1 infection (PHI) and of two antiretroviral therapies [highly active antiretroviral therapy (HAART) or reverse transcriptase inhibitors (RTI)] on activation, differentiation and survival of B cells., Methods: Naive and memory B cells from three groups [PHI (31), chronic infection (26) and healthy donors (12)] were studied for surface expression of Fas, LAIR-1, CD70, intracellular expression of Bcl-2 and spontaneous apoptosis. Fluorescence activated cell sorting (IgD+IgM+CD19+CD27+) and short-term cell culture to analyse induction of CD25 on B cells were performed in five patients with PHI. Patients with PHI were sampled at baseline, and after 1 and 6 months of therapy. Results were analysed by parametric and non-parametric tests and by mathematical modelling., Results: In PHI, B cells were significantly decreased; naive and memory B lymphocytes showed a high degree of activation, manifested by hypergammaglobulinaemia, altered expression of Fas and LAIR-1, and high rate of spontaneous apoptosis. Antiretroviral treatment improved the activation/differentiation status of B cells, reduced apoptosis to levels comparable to those in healthy individuals and restored the ability of B cells to respond to T cell-dependent activation. B cells showed slightly better recovery in patients taking HAART than in those taking RTI. Decreased IgM-positive memory B cells and lower induction of CD25 expression on B cells upon T cell activation at diagnosis of PHI was shown in five patients tested. These parameters normalized after 6 months of therapy., Conclusion: B cell dysfunctions found in chronic HIV-1 infection appear during PHI and initiation of antiretroviral therapy early during infection may help to preserve the B cell compartment.

The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+ cells from both humans and untreated mice, (iii) inhibit Mip-1beta chemotaxis of CD4+ CCR5+ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

Unconventional immune responses have been demonstrated in individuals who, despite repeated exposure to human immunodeficiency virus (HIV) infection, remain seronegative. As environmental exposure to pathogens and genetic background may modulate immune responses differentially, one Italian and two Asian populations of HIV-1-exposed seronegative individuals were studied. In serum samples from each group, IgG to CCR5, IgG to CD4 and IgA to gp41 were measured, which were previously described as markers of unconventional immunity in HIV-exposed seronegative Caucasians. Given the importance of conformational epitopes in virus-cell interactions, IgG to CD4-gp120 complex was also measured. It was found that markers of HIV exposure were present in all populations studied. HIV-specific humoral responses (IgA to gp41 and IgG to CD4-gp120 complex) were extremely significant predictors of HIV exposure (P<0.0001 in both cases), whereas the predictive values of anti-cell antibodies (anti-CCR5 and anti-CD4) varied between populations. Evidence is provided for the correlation of these differences with route of exposure to HIV and level of natural antibodies to cross-reactive microbial antigens. In conclusion, exposed seronegative individuals of ethnically different origins display similar signs of HIV-dependent unconventional immunity. A specific relevance must be attributed to different innate and acquired factors.

More than two decades since its discovery, human immunodeficiency virus (HIV) epidemic is still a major burden for health, social and economical reasons on all over the world. Despite the huge effort in basic and applied research, aimed to control virus spread and to design effective therapeutic strategies, an HIV vaccine is not available yet and current therapeutics approaches cannot prevent the infection. To date, both host genetic repertoire, innate and acquired immune responses, viral mutation or attenuation have been invoked to explain the higher or lower individual susceptibility to the infection. The existence of some people somewhat "immune" from infection, despite dealing with repeated HIV exposure, as well as the extremely slow disease progression in some HIV infected individuals, offers valuable clues to elucidate mechanisms underlying natural HIV resistance. Strikingly, both such cohorts, the so-called Exposed Seronegative (ESN, EU, HEPS) and the Slow Progressor (SP, LTNP, LTS) individuals have common immune responses, e.g. the generation of neutralising antibodies directed against common targets, which can play a protective role in virus entry and/or spread. This review focuses on naturally occurring humoral responses to HIV exposure/infection. Moreover, whether such antibodies are induced in response to a peculiar scenario of HIV infection or are generated in the context of an individual innate mode to clear virus infection is a puzzling question, which will be addressed here. The comprehension of mechanisms of natural resistance to HIV infection may have implications for the identification of anti-viral novel strategies and in particular for the development of innovative diagnostics, therapeutics and vaccine design.

Background: Despite multiple, repeated exposures to HIV-1, some individuals never seroconvert. Mucosal and systemic immune correlates of this condition have been analysed in HIV-1-exposed women but no data are available concerning mucosal immunity and HIV-1-specific immune responses in exposed but uninfected men., Design: We analysed cellular and humoral immune parameters in peripheral lymphocytes, seminal fluid and urethral swabs of 14 recently HIV-1-exposed seonegative (ESN) heterosexual men, seven HIV-seropositive patients and seven healthy controls., Results: HIV-1-specific IgA were detected in urethral swabs of 11 out of 14 ESN and of six out of seven HIV-seropositive patients; Env- and Gag-specific IFNgamma-producing CD4 and CD8 peripheral lymphocytes were present in ESN and HIV-seropositive patients; seminal lymphocytes, but not peripheral blood lymphocytes, of ESN were enriched in activated populations (CD8CD38RO and CD4CD25). p24-specific cytotoxic T lymphocytes were correlated with the percentage of CD4 in the HIV-seropositive partners. High urethral concentrations of HIV-1-specific IgA were seen in those ESN with the most recent unprotected sexual episode., Conclusions: This is the first report of HIV-specific mucosal immunity in ESN men. These data add to the body of knowledge of the immune correlates present in exposed, uninfected individuals and might be important in vaccine design.

Objective: To study the effect of highly active antiretroviral therapy (HAART) with and without hydroxyurea (HU) on changes in plasma viral load (VL) set-point, and on HIV-1-specific responses, after five cycles of structured treatment interruptions (STI)., Methods: A group of 20 patients taking HAART for chronic HIV infection with VL < 20 copies/ml were randomized to continue HAART or HAART plus HU for 24 weeks followed by five STI cycles. HU was also stopped in cycles 1-3 but continued in cycles 4 and 5. The number of individuals maintaining a VL set-point < 5000 copies/ml during the fifth interruption were determined., Results: VL remained < 5000 copies/ml in eight out of nine patients in the HU group and in four out of ten patients in the HAART group after a median 48 weeks of follow-up after the fifth interruption ( P=0.039). By STI cycle 5, there was a significant increase in the neutralizing activity (NA), in both magnitude and breadth of the total cytotoxic T lymphocyte (CTL) response and in lymphoproliferative response (LPR) from baseline. No significant differences were observed between HAART and HU groups in NA, CTL and LPR at any time-point. There were no differences in the NA titers at any time-point between responder and non-responder patients. There was a trend for higher CTL and LPR levels in responder patients (P= 0.10)., Conclusions: In this randomized, controlled study of STI with cycles of HAART or HAART plus HU, a lower peak VL rebound and a lower VL set-point was achieved in patients continuing HU while other drugs were discontinued. HU did not blunt anti-HIV-1-specific responses; however, control of VL did not correlate with anti-HIV-1-specific cellular immune responses.

Background: HIV-specific IgA is present in HIV-exposed uninfected individuals (EU) and neutralizes primary strains of HIV-1 in vitro., Objectives: To analyse the antigenic correlates of HIV-1 neutralization using HIV epitopes and IgA from EU and HIV-seropositive individuals., Methods: Sera from six heterosexual couples discordant for HIV serostatus, six age-matched HIV-infected subjects and six healthy controls (HC; as negative controls) were analysed. IgA binding on HIV Env recombinant proteins was assayed. Serum IgA was affinity purified on specific Env peptides and tested in HIV neutralization using resting and activated peripheral blood mononuclear cells as target. Monoclonal antibody 2F5 was used as neutralizing positive control. BALB/c mice were immunized with specific gp41 peptide and anti-sera were tested in syncytia formation and in HIV viral replication., Results: IgA of EU exclusively bound an epitope within gp41; this epitope was restricted to residues 582-588 (QARILAV) and corresponded to the leucine zip motif in the alpha-helical region. IgA of HIV-positive patients recognized epitopes expressed both in gp120 and gp41; these epitopes were in the N-terminal portion of the extramembrane region. Additionally, IgA of EU and antisera of QARILAV-immunized Balb/C mice blocked syncytia formation and viral replication. The dose-dependent neutralization behaviour of specific QARILAV-purified IgA was very similar to that obtained with monoclonal antibody 2F5., Conclusion: These results have important implications for the development of vaccines and therapeutical strategies against HIV infection., (Copyright 2002 Lippincott Williams & Wilkins)

There is an urgent need for a universally effective HIV-1 vaccine, but whether a vaccine will be able to protect against HIV-1 of different clades is a significant concern. IgA from HIV-1-exposed, persistently seronegative (HEPS) subjects has been shown to neutralize HIV-1 and to block epithelial HIV-1 transcytosis, and it may target novel HIV-1 epitopes. We have tested the ability of plasma and mucosal IgA purified from HEPS subjects to neutralize HIV-1 primary isolates of different viral clades and phenotypes. IgA from two groups of HEPS subjects was tested: sex workers from Nairobi, Kenya, where clades A and D predominate, and the heterosexual partners of individuals infected by clade B virus. HIV-1-infected and low-risk uninfected individuals were included as controls. IgA purified from the blood, genital tract, and saliva of most HEPS sex workers demonstrated significant cross-clade HIV-1 neutralization, whereas a more clade-restricted pattern of neutralization was found in partners of clade B-infected individuals. IgA purified from HIV-1-infected individuals also mediated cross-clade neutralization, whereas IgA from uninfected controls lacked neutralizing activity. In conclusion, mucosal and plasma IgA from HEPS subjects neutralizes HIV-1 of different clades. This ability to induce HIV-1-specific systemic and mucosal IgA may be an important feature of an effective prophylactic HIV-1 vaccine.

Objectives: Some individuals remain uninfected despite repeated exposure to HIV-1 [exposed-uninfected (EU)]. In addition to genetic factors, acquired immune responses elicited by repeated exposure to HIV antigens may contribute to protection. We investigated the ability of unstimulated CD8+ T lymphocytes from EU individuals to inhibit HIV-1 infection., Methods: Peripheral blood CD8+ T lymphocytes from a well-characterized cohort of 16 HIV-1-discordant monogamous heterosexual couples were tested for their suppressive activity against HIV-1 strains displaying different coreceptor usage (R5, X4, X4R5). To evaluate the in vivo functional competence of CD8+ T cells, no ex vivo activatory stimuli were used prior to cocultivation with infected CD4+ T cells. In some experiments, a semi-permeable membrane was used to separate CD4+ and CD8+ T cells., Results: Unstimulated CD8+ T cells from all but one of the EU individuals analysed effectively inhibited the growth of all HIV-1 strains, regardless of their coreceptor usage, with a mean potency similar to that of asymptomatic HIV-infected patients. The HIV-inhibitory activity persisted for a long time after ceasing high-risk sexual behaviour, although a moderate decline was observed starting 4 years after the last risk episode. Transwell culture experiments showed that soluble factors are involved in CD8-mediated viral suppression, although the activity was higher when cell-to-cell contact was allowed., Conclusions: These data demonstrate that CD8+ T cells from EU individuals exert a strong, broad-spectrum HIV-suppressive activity, suggesting a role of non-cytotoxic antiviral mechanisms in resistance to HIV-1 infection.

The distribution of Human Immunodeficiency Virus type 1 (HIV-1) clades is evaluated in primary HIV-1 infections (PHIs) occurring through sexual transmission in Lombardia, the Italian region with the highest prevalence/incidence of HIV-1 infections. The two primary inclusion parameters for enrollment were sexual transmission and < 1 year seroconversion. Thirty-four enrolled patients have been analysed so far at the molecular level, to characterize their infecting HIV-1 population. Two HIV-1 genomic regions with different rates of genetic variability, the hypervariable C2-V3 fragment of the env gene and the conserved 5' end of the gag p17, were amplified by Polymerase Chain Reaction (PCR) in peripheral blood mononuclear cells (PBMCs) and characterized by direct DNA sequence analysis. Pairwise nucleotide alignment and phylogenetic analyses show that, although with a high range of nucleotide variability, 32 out of the 34 HIV-1 isolates identified in this PHI cohort fall under the clade B genotype. The two remaining isolates, detected in a couple formed by a Nigerian woman and her Italian partner, consistently cluster with clade G standards in both sub-genomic regions. The amino acid sequences confirm this classification, showing clade-specific residues both in the V3 and p17 regions. These data suggest that the B clade is still prevalently associated with acute primary HIV-1 infections occurring in Italy through sexual transmission. However, the significant intra-clade variability and the identification of non-B clades strongly indicate the relevance of continuous molecular monitoring of the HIV-1 isolates circulating in Italy, for prognostic evaluations as well as preventive and therapeutic strategies.

The effect of Highly Active Antiretroviral Therapy (HAART) on binding and neutralizing antibody responses to human immunodeficiency virus type-1 (HIV-1) during primary and chronic infection was investigated. Seven patients HAART treated during primary infection, six HAART treated during chronic infection and five patients treated only with ZVD (Zidovudine) were analysed. HAART inhibited the development of anti env antibodies during primary infection. Administering HAART during primary infection usually did not substantially affect the development of weak neutralizing antibody responses against autologous virus. However, we demonstrated that very early treatment, during seroconversion, induce in some cases, a strong neutralizing antibodies against autologous virus. These results may be relevant for understanding how HAART may elicit a strong protective responses and may be useful in developing new strategies designed to achieve a long term control of the HIV infection.

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.

Background: HIV infection in Africa is associated with immune activation and a cytokine profile that stimulates CCR5 expression. We investigated whether this immune activation is environmentally driven; if a dominant expression of CCR5 could indeed be detected in African individuals; and if R5 HIV strains would be prevalent in this population., Methods: Freshly drawn peripheral blood mononuclear cells from HIV-uninfected African and Italian individuals living in rural Africa, from HIV-uninfected Africans and Italians living in Italy, and from HIV-infected African and Italian patients were analysed. Determinations of HIV coreceptor-specific mRNAs and immunophenotype analyses were performed in all samples. Virological analyses included virus isolation and characterization of plasma neutralizing activity., Findings: Results showed that: immune activation is detected both in Italian and African HIV-uninfected individuals living in Africa but not in African subjects living in Italy; CCR5-specific mRNA is augmented and the surface expression of CCR5 is increased in African compared with Italian residents (CXCR4-specific mRNA is comparable); R5-HIV strains are isolated prevalently from lymphocytes of African HIV-infected patients; and plasma neutralizing activity in HIV-infected African patients is mostly specific for R5 strains., Conclusions: Immune activation in African residents is environmentally driven and not genetically predetermined. This immune activation results in a skewing of the CCR5 : CXCR4 ratio which is associated with a prevalent isolation of R5 viruses. These data suggest that the selection of the predominant virus strain within the population could be influenced by an immunologically driven pattern of HIV co receptor expression.

Objective: To characterize functional properties of HIV-specific IgA in samples representing both systemic and mucosal compartments of HIV-1 highly exposed persistently seronegative (HEPS) individuals., Methods: IgA was purified from plasma and mucosal samples from HEPS individuals and tested for the ability to neutralize infection of peripheral blood mononuclear cells (PBMC) by a non-syncytium inducing HIV-1 (clade B) primary isolate. None of these individuals had measurable HIV-1-specific IgG., Results: HIV-1-specific neutralizing activity of the purified IgA from plasma (n = 15), saliva (n = 15) and cervicovaginal fluid (CVF) (n = 14) were found in the majority of samples (73, 73 and 79%, respectively). In contrast, plasma, saliva and CVF samples of low-risk, uninfected HIV-seronegative individuals lacked neutralizing IgA, with the exception of two out of 34 (6%) saliva samples., Conclusion: Mucosal and plasma IgA from HEPS individuals can neutralize HIV-1 infection.

Exposure to HIV does not necessarily result in infection. Because primary HIV infection is associated with CCR5-tropic HIV variants (R5), CCR5-specific Abs in the sera of HIV-seronegative, HIV-exposed individuals (ESN) might be associated with protection against infection. We analyzed sera from ESN, their HIV-infected sexual partners (HIV+), and healthy controls (USN) searching for CCR5-specific Abs, studying whether incubation of PBMC with sera could prevent macrophage inflammatory protein 1 beta (Mip1 beta) (natural ligand of CCR5) binding to CCR5. Results showed that Mip1 beta binding to CCR5 was not modified by sera of either 40 HIV+ or 45 USN but was greatly reduced by sera of 6/48 ESN. Binding inhibition was due to Abs reactive with CCR5. The CCR5-specific Abs neutralized the infectivity of primary HIV isolates obtained from the corresponding HIV+ partners and of R5-primary HIV strains, but not that of CXCR4-tropic or amphitropic HIV strains. Immunoadsorption on CCR5-transfected, but not on CXCR4-transfected, cells removed CCR5-specific and virus-neutralizing Abs. Epitope mapping on purified CCR5-specific Abs showed that these Abs recognize a conformational epitope in the first cysteine loop of CCR5 (aa 89-102). Affinity-purified anti-CCR5-peptide neutralized the infectivity of R5 strains of HIV-1. Anti-CCR5 Abs inhibited Mip1beta-induced chemotaxis of PBMC from healthy donors. PBMC from two ESN (with anti-CCR5 Abs) were CCR5-negative and could not be stimulated by Mip1beta in chemotaxis assays. These results contribute to clarifying the phenomenon of immunologic resistance to HIV and may have implications for the development of a protective vaccine.

Despite repeated exposures to HIV-1, some individuals remain seronegative. This study reports that sera from a fraction of exposed seronegative (ESN) subjects showed HIV-neutralizing activity; 5 of 17 ESN sera and none of 17 controls neutralized two different HIV-1 primary isolates (range of neutralizing titers: 1/20 to 1/60). The neutralizing activity was associated with the IgG fraction of 4 of 4 neutralizing ESN sera. Moreover, in 11 of 17 and 9 of 17 ESN sera (but none of the control sera) we found antibodies against HLA class I and CD4, respectively. One of the ESN sera (EU22) neutralized efficiently the primary virus derived from the seropositive partner and showed a good broadly cross-reactive neutralization. Immunoadsorption of two IgG fractions from EU19 and EU22 on peripheral blood mononuclear cells (PBMC) removed virus-neutralizing antibodies. The correlations between the ESN status and neutralizing activity (p<0.05), anti-HLA antibodies (p<0.0002), and anti-CD4 antibodies (p<0.001) were statistically significant. However, there was no statistically significant correlation between neutralizing activity and either anti-HLA or anti-CD4 antibodies. It can therefore be said that exposure to HIV-1 without seroconversion is, in some individuals, associated with HIV-neutralizing antibodies (not directed against viral antigens) and/or with anti-cell autoantibodies, which are possibly specific for cellular antigens involved in the infection/entry process.

Human immunodeficiency virus (HIV)-specific IgA can be detected in cervical secretions, saliva, and sera of HIV-infected and HIV-uninfected individuals with a known exposure to the virus. IgA from HIV-uninfected exposed seronegative individuals (ESN) neutralize in vitro primary strains of HIV-1. We analyzed the epitopes of HIV recognized by serum HIV-specific IgA of ESN individuals to identify the antigenic correlates of HIV neutralization in exposed-uninfected subjects, and to verify whether different epitopes would be recognized by HIV-specific IgA of ESN and of HIV-infected patients. Results confirmed that HIV-neutralizing IgA are detected in sera of ESN and showed that neutralization of primary HIV strains is mediated by the recognition of different epitopes in HIV-infected patients and ESN. Thus, whereas IgA of HIV+ individuals recognize epitopes expressed both within gp120 and gp41, IgA of ESN exclusively bind to gp41-expressed epitopes. Epitope mapping revealed that the epitope recognized by serum IgA of ESN on gp41 is restricted to aa 581-584 (LQAR) and corresponds to coiled coil pocket in the alpha helic region. In contrast, the epitope seen by IgA of HIV-infected patients on gp41 is identified by two regions; the first is contained within the cystein loop (aa 589-618), the second correspond to C terminal region in the extra membrane region of gp 41 (aa 642-673). Thus, we have identified and characterized the epitopes that mediate neutralization of HIV in individuals in whom infection does not occur despite multiple exposures to the virus. These results have important implications for the development of a new therapy against HIV infection.

We investigate the effects of highly active antiretroviral therapy (HAART) on humoral immune responses during a 24-month follow up of 15 HIV patients with acute primary HIV infection. The patients were divided into three groups on the basis of the therapeutic protocol they were following at the time of entry: a) five naive patients (untreated or treated with only ZDV or AZT); b) five patients following a triple combination of ZDV+ lamivudine (3TC)+ saquinovir (SQV); and c) five patients on a four-drug combination of ZDV+3TC+SQV+ ritonavir (RTV). The results show that the early introduction of HAART greatly reduces plasma viremia levels and restores the number of CD4 cells. A significant correlation was found between anti HIV neutralising activity and the four-drug, but not the three-drug combination. The reduction in infectivity was directed against viruses of different clades and associated with immunoglobulin fractions. Moreover, the neutralising antibodies in the HAART-treated patients appeared after two weeks of treatment and remained stable throughout the 24 months of follow up. The early appearance of neutralising antibodies represent an important component of immune responses during primary HIV infection, may contribute towards immune reconstitution in patients on HAART, and give further information that may be useful in developing new strategies designed to eradicate the disease.