Cooperation Between Systemic and Mucosal Antibodies Induced by Virosomal Vaccines Targeting HIV-1 Env: Protection of Indian Rhesus Macaques Against Low-Dose Intravaginal SHIV Challenges.

A virosomal vaccine inducing systemic/mucosal anti-HIV-1 gp41 IgG/IgA had previously protected Chinese-origin rhesus macaques (RMs) against vaginal SHIVSF162P3 challenges. Here, we assessed its efficacy in Indian-origin RMs by intramuscular priming/intranasal boosting (n=12/group). Group K received virosome-P1-peptide alone (harboring the Membrane Proximal External Region), Group L combined virosome-rgp41 plus virosome-P1, and Group M placebo virosomes. Vaccination induced plasma binding but no neutralizing antibodies. Five weeks after boosting, all RMs were challenged intravaginally with low-dose SHIVSF162P3 until persistent systemic infection developed. After SHIV challenge #7, six controls were persistently infected versus only one Group L animal (vaccine efficacy 87%; P=0.0319); Group K was not protected. After a 50% SHIV dose increase starting with challenge #8, protection in Group L was lost. Plasmas/sera were analyzed for IgG phenotypes and effector functions; the former revealed that protection in Group L was significantly associated with increased binding to FcγR2/3(A/B) across several time-points, as were some IgG measurements. Vaginal washes contained low-level anti-gp41 IgGs and IgAs, representing a 1-to-5-fold excess over the SHIV inoculum's gp41 content, possibly explaining loss of protection after the increase in challenge-virus dose. Virosomal gp41-vaccine efficacy was confirmed during the initial seven SHIV challenges in Indian-origin RMs when the SHIV inoculum had at least 100-fold more HIV RNA than acutely infected men's semen. Vaccine protection by virosome-induced IgG and IgA parallels the cooperation between systemically administered IgG1 and mucosally applied dimeric IgA2 monoclonal antibodies that as single-agents provided no/low protection - but when combined, prevented mucosal SHIV transmission in all passively immunized RMs.

Mucosal AIDS virus transmission is enhanced by antiviral IgG isolated early in infection.

OBJECTIVE: Antibody-dependent enhancement (ADE) affects host-virus dynamics in fundamentally different ways: i) enhancement of initial virus acquisition, and/or ii) increased disease progression/severity. Here we address the question whether anti-HIV-1 antibodies can enhance initial infection. While cell-culture experiments hinted at this possibility, in-vivo proof remained elusive. DESIGN: We used passive immunization in nonhuman primates challenged with simian-human immunodeficiency virus (SHIV), a chimera expressing HIV-1 envelope. We purified IgG from rhesus monkeys with early-stage SHIV infection - before cross-neutralizing anti-HIV-1 antibodies had developed - and screened for maximal complement-mediated antibody-dependent enhancement (C'-ADE) of viral replication with a SHIV strain phylogenetically distinct from that harbored by IgG donor macaques. IgG fractions with maximal C'-ADE but lacking neutralization were combined to yield enhancing anti-SHIV IgG (enSHIVIG). RESULTS: We serially enrolled naive macaques (Group 1) to determine the minimal and 50% animal infectious doses required to establish persistent infection after intrarectal SHIV challenge. The first animal was inoculated with a 1 : 10 virus-stock dilution; after this animal's viral RNA load was >104copies/ml, the next macaque was challenged with 10x less virus, a process repeated until viremia no longer ensued. Group 2 was pretreated intravenously with enSHIVIG 24 h before SHIV challenge. Overall, Group 2 macaques required 3.4-fold less virus compared to controls (P = 0.002). This finding is consistent with enhanced susceptibility of the passively immunized animals to mucosal SHIV challenge. CONCLUSION: These passive immunization data give proof of IgG-mediated enhanced virus acquisition after mucosal exposure - a potential concern for antibody-based AIDS vaccine development.

Adaptation of an R5 Simian-Human Immunodeficiency Virus Encoding an HIV Clade A Envelope with or without Ablation of Adaptive Host Immunity: Differential Selection of Viral Mutants.

Simian-human immunodeficiency virus (SHIV) infection in rhesus macaques (RMs) resembles human immunodeficiency virus type 1 (HIV-1) infection in humans and serves as a tool to evaluate candidate AIDS vaccines. HIV-1 clade A (HIV-A) predominates in parts of Africa. We constructed an R5 clade A SHIV (SHIV-A; strain SHIV-KNH1144) carrying env from a Kenyan HIV-A. SHIV-A underwent rapid serial passage through six RMs. To allow unbridled replication without adaptive immunity, we simultaneously ablated CD8+ and B cells with cytotoxic monoclonal antibodies in the next RM, resulting in extremely high viremia and CD4+ T-cell loss. Infected blood was then transferred into two non-immune-depleted RMs, where progeny SHIV-A showed increased replicative capacity and caused AIDS. We reisolated SHIV-KNH1144p4, which was replication competent in peripheral blood mononuclear cells (PBMC) of all RMs tested. Next-generation sequencing of early- and late-passage SHIV-A strains identified mutations that arose due to "fitness" virus optimization in the former and mutations exhibiting signatures typical for adaptive host immunity in the latter. "Fitness" mutations are best described as mutations that allow for better fit of the HIV-A Env with SIV-derived virion building blocks or host proteins and mutations in noncoding regions that accelerate virus replication, all of which result in the outgrowth of virus variants in the absence of adaptive T-cell and antibody-mediated host immunity.IMPORTANCE In this study, we constructed a simian-human immunodeficiency virus carrying an R5 Kenyan HIV-1 clade A env (SHIV-A). To bypass host immunity, SHIV-A was rapidly passaged in naive macaques or animals depleted of both CD8+ and B cells. Next-generation sequencing identified different mutations that resulted from optimization of viral replicative fitness either in the absence of adaptive immunity or due to pressure from adaptive immune responses.

Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism.

The phase III RV144 human immunodeficiency virus (HIV) vaccine trial conducted in Thailand remains the only study to show efficacy in decreasing the HIV acquisition risk. In Thailand, circulating recombinant forms of HIV clade A/E (CRF01_AE) predominate; in such viruses, env originates from clade E (HIV-E). We constructed a simian-human immunodeficiency virus (SHIV) chimera carrying env isolated from an RV144 placebo recipient in the SHIV-1157ipd3N4 backbone. The latter contains long terminal repeats (LTRs) with duplicated NF-κB sites, thus resembling HIV LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV-E1, to rhesus macaques: the simultaneous depletion of B and CD8+ cells followed by the intramuscular inoculation of proviral DNA and repeated administrations of cell-free virus. High-level viremia and CD4+ T-cell depletion ensued. Passage 3 virus unexpectedly caused acute, irreversible CD4+ T-cell loss; the partially adapted SHIV had become dual tropic. Virus and IMCs with exclusive R5 tropism were reisolated from earlier passages, combined, and used to complete adaptation through additional macaques. The final isolate, SHIV-E1p5, remained solely R5 tropic. It had a tier 2 neutralization phenotype, was mucosally transmissible, and was pathogenic. Deep sequencing revealed 99% Env amino acid sequence conservation; X4-only and dual-tropic strains had evolved independently from an early branch of parental SHIV-E1. To conclude, our primate model data reveal that SHIV-E1p5 recapitulates important aspects of HIV transmission and pathobiology in humans.IMPORTANCE Understanding the protective principles that lead to a safe, effective vaccine against HIV in nonhuman primate (NHP) models requires test viruses that allow the evaluation of anti-HIV envelope responses. Reduced HIV acquisition risk in RV144 has been linked to nonneutralizing IgG antibodies with a range of effector activities. Definitive experiments to decipher the mechanisms of the partial protection observed in RV144 require passive-immunization studies in NHPs with a relevant test virus. We have generated such a virus by inserting env from an RV144 placebo recipient into a SHIV backbone with HIV-like LTRs. The final SHIV-E1p5 isolate, grown in rhesus monkey peripheral blood mononuclear cells, was mucosally transmissible and pathogenic. Earlier SHIV-E passages showed a coreceptor switch, again mimicking HIV biology in humans. Thus, our series of SHIV-E strains mirrors HIV transmission and disease progression in humans. SHIV-E1p5 represents a biologically relevant tool to assess prevention strategies.

Defense-in-depth by mucosally administered anti-HIV dimeric IgA2 and systemic IgG1 mAbs: complete protection of rhesus monkeys from mucosal SHIV challenge.

Although IgA is the most abundantly produced immunoglobulin in humans, its role in preventing HIV-1 acquisition, which occurs mostly via mucosal routes, remains unclear. In our passive mucosal immunizations of rhesus macaques (RMs), the anti-HIV-1 neutralizing monoclonal antibody (nmAb) HGN194, given either as dimeric IgA1 (dIgA1) or dIgA2 intrarectally (i.r.), protected 83% or 17% of the RMs against i.r. simian-human immunodeficiency virus (SHIV) challenge, respectively. Data from the RV144 trial implied that vaccine-induced plasma IgA counteracted the protective effector mechanisms of IgG1 with the same epitope specificity. We thus hypothesized that mucosal dIgA2 might diminish the protection provided by IgG1 mAbs targeting the same epitope. To test our hypothesis, we administered HGN194 IgG1 intravenously (i.v.) either alone or combined with i.r. HGN194 dIgA2. We enrolled SHIV-exposed, persistently aviremic RMs protected by previously administered nmAbs; RM anti-human IgG responses were undetectable. However, low-level SIV Gag-specific proliferative T-cell responses were found. These animals resemble HIV-exposed, uninfected humans, in which local and systemic cellular immune responses have been observed. HGN194 IgG1 and dIgA2 used alone and the combination of the two neutralized the challenge virus equally well in vitro. All RMs given only i.v. HGN194 IgG1 became infected. In contrast, all RMs given HGN194 IgG1+dIgA2 were completely protected against high-dose i.r. SHIV-1157ipEL-p challenge. These data imply that combining suboptimal defenses at the mucosal and systemic levels can completely prevent virus acquisition. Consequently, active vaccination should focus on defense-in-depth, a strategy that seeks to build up defensive fall-back positions well behind the fortified frontline.

Passive immunization of macaques with polyclonal anti-SHIV IgG against a heterologous tier 2 SHIV: outcome depends on IgG dose.

BACKGROUND: A key goal for HIV-1 envelope immunogen design is the induction of cross-reactive neutralizing antibodies (nAbs). As AIDS vaccine recipients will not be exposed to strains exactly matching any immunogens due to multiple HIV-1 quasispecies circulating in the human population worldwide, heterologous SHIV challenges are essential for realistic vaccine efficacy testing in primates. We assessed whether polyclonal IgG, isolated from rhesus monkeys (RMs) with high-titer nAbs (termed SHIVIG), could protect RMs against the R5-tropic tier-2 SHIV-2873Nip, which was heterologous to the viruses or HIV-1 envelopes that had elicited SHIVIG. RESULTS: SHIVIG demonstrated binding to HIV Gag, Tat, and Env of different clades and competed with the broadly neutralizing antibodies b12, VRC01, 4E10, and 17b. SHIVIG neutralized tier 1 and tier 2 viruses, including SHIV-2873Nip. NK-cell depletion decreased the neutralizing activity of SHIVIG 20-fold in PBMC assays. Although SHIVIG neutralized SHIV-2873Nip in vitro, this polyclonal IgG preparation failed to prevent acquisition after repeated intrarectal low-dose virus challenges, but at a dose of 400 mg/kg, it significantly lowered peak viremia (P = 0.001). Unexpectedly, single-genome analysis revealed a higher number of transmitted variants at the low dose of 25 mg/kg, implying increased acquisition at low SHIVIG levels. In vitro, SHIVIG demonstrated complement-mediated Ab-dependent enhancement of infection (C'-ADE) at concentrations similar to those observed in plasmas of RMs treated with 25 mg/kg of SHIVIG. CONCLUSION: Our primate model data suggest a dual role for polyclonal anti-HIV-1 Abs depending on plasma levels upon virus encounter.

Isolation of monoclonal antibodies with predetermined conformational epitope specificity.

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.

Gonadotropin-releasing hormone/human chorionic gonadotropin beta based recombinant antibodies and vaccines.

Gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) are unique targets for the control of fertility. Immunological approaches to neutralizing these hormones have additional utility in cancer treatment. Vaccines have been developed against both GnRH and hCG and these have undergone Phase I/II clinical trials documenting their safety, reversibility and efficacy. The heterospecies dimer hCG vaccine prevented pregnancy in women of proven fertility without impairment of ovulation or derangement of menstrual regularity and bleeding profiles. The protective threshold of antibody titers to achieve efficacy was determined in these first-ever trials. Recently, a recombinant vaccine against the beta subunit of hCG linked to the B subunit of heat labile enterotoxin has been made and expressed as a glycosylated conjugate in Pichia pastoris. Experiments indicate its ability to generate antibodies above the protective threshold in all immunized Balb/c mice. Ectopic expression of hCG/hCGbeta is observed in many advanced stage cancers of various origins. A chimeric high affinity and specific recombinant antibody against hCGbeta linked to curcumin kills hCGbeta expressing T lymphoblastic leukemia cells without any deleterious effect. Several synthetic and recombinant vaccines have been developed against GnRH. These reduce serum testosterone to castration levels causing atrophy of the prostate. Three Phase I/II clinical trials conducted in India and Austria have shown that these vaccines elicit non-surgical reduction of testosterone, a fall in prostate specific antigen and clinical improvement of prostate carcinoma patients. A multimer recombinant vaccine against GnRH has high efficacy for sterilization of pigs and other animals.

Selective killing of leukemia and lymphoma cells ectopically expressing hCGbeta by a conjugate of curcumin with an antibody against hCGbeta subunit.

OBJECTIVE: A variety of cancers ectopically express human chorionic gonadotropin beta (hCGbeta). Patients harboring such cancers have poor prognosis and adverse survival. A recombinant chimeric antibody, cPiPP, exhibiting high affinity and specificity for hCGbeta/hCG was engineered. This study was designed to determine whether this antibody alone or conjugated to curcumin can selectively kill tumor cells expressing hCGbeta. EXPERIMENTAL DESIGN: The study was carried out on MOLT-4 and U-937 cells expressing hCGbeta and on peripheral blood leukocytes of acute myeloid leukemia (AML) patients. The anticancerous compound curcumin was conjugated to cPiPP. The binding of cPiPP and cPiPP-curcumin conjugate to cells was studied by flow cytometry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), FACS with propidium iodide staining, trypan blue exclusion assay and microscopy. RESULTS: The antibody did not impair the growth of MOLT-4 and U-937 cells in culture. Its conjugate with curcumin, however, was lethal to both cell lines. The immunoconjugate killed tumor cells bearing the CD33 marker of an AML patient expressing hCGbeta but did not have a similar action on cells of another AML patient with the CD13 marker but who was negative for hCGbeta. CONCLUSION: A humanized antibody against hCGbeta linked to curcumin has potential for therapy of hCGbeta-expressing tumors.

Recent advances in contraceptive vaccine development: a mini-review.

Contraceptive vaccines (CV) may provide viable and valuable alternatives to the presently available methods of contraception. The molecules that are being explored for CV development either target gamete production [luteinizing hormone-releasing hormone (LHRH)/GnRH, FSH], gamete function [sperm antigens and oocyte zona pellucida (ZP)], and gamete outcome (HCG). CV targeting gamete production have shown varied degrees of efficacy; however, they either affect sex steroids causing impotency and/or show only a partial rather than a complete effect in inhibiting gametogenesis. However, vaccines based on LHRH/GnRH are being developed by several pharmaceutical companies as substitutes for castration of domestic pets, farm and wild animals, and for therapeutic anticancer purposes such as in prostatic hypertrophy and carcinoma. These vaccines may also find applications in clinical situations that require the inhibition of increased secretions of sex steroids, such as in uterine fibroids, polycystic ovary syndrome, endometriosis and precocious puberty. CV targeting molecules involved in gamete function such as sperm antigens and ZP proteins are exciting choices. Sperm constitute the most promising and exciting target for CV. Several sperm-specific antigens have been delineated in several laboratories and are being actively explored for CV development. Studies are focused on delineating appropriate sperm-specific epitopes, and increasing the immunogenicity (specifically in the local genital tract) and efficacy on the vaccines. Anti-sperm antibody (ASA)-mediated immunoinfertility provides a naturally occurring model to indicate how a vaccine might work in humans. Vaccines based on ZP proteins are quite efficacious in producing contraceptive effects, but may induce oophoritis, affecting sex steroids. They are being successfully tested to control feral populations of dogs, deer, horses and elephants, and populations of several species of zoo animals. The current research for human applicability is focused on delineating infertility-related epitopes (B-cell epitopes) from oophoritis-inducing epitopes (T-cell epitopes). Vaccines targeting gamete outcome primarily focus on the HCG molecule. The HCG vaccine is the first vaccine to undergo Phase I and II clinical trials in humans. Both efficacy and lack of immunopathology have been reasonably well demonstrated for this vaccine. At the present time, studies are focused on increasing the immunogenicity and efficacy of the birth control vaccine, and examining its clinical applications in various HCG-producing cancers. The present article will focus on the current status of the anti-sperm, anti-ZP, anti-LHRH/GnRH and anti-HCG vaccines.