Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection

Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50-70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV-vpu+ (refs. 5,6), a chimeric simian-human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV-vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV-vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV-vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.     

Neutralizing Monoclonal Antibodies Block Human Immunodeficiency Virus Type 1 Infection of Dendritic Cells and Transmission to T Cells

Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14⁺ blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3⁺ T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1.     

Characterization of human class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12

We have previously generated human monoclonal anti-human immunodeficiency virus type 1 (anti-HIV-1) antibodies 2F5IgG and 2G12IgG with an exceptional cross-clade neutralizing potential. 2F5IgG and 2G12IgG passively administrated to macaques were able to confer complete protection from both intravenous and mucosal challenge with pathogenic HIV-simian immunodeficiency virus chimeric strains and have shown beneficial effects in a phase-1 clinical trial. We now class-switched 2F5 and 2G12 to the immunoglobulin M (IgM) or IgA isotype, to enforce features like avidity, complement activation, or the potential to neutralize mucosal transmission. For this purpose we expressed functional polymeric 2F5 and 2G12 antibodies in CHO cells and evaluated their anti-HIV-1 activity in vitro. The class switch had a strong impact on the protective potential of 2F5 and 2G12. 2G12IgM inhibited HIV-1 infection of peripheral blood mononuclear cell cultures up to 28-fold-more efficiently than the corresponding IgG and neutralized all of the primary isolates tested. The 2F5 and 2G12 antibodies of all isotypes were able to interact with active human serum to inhibit viral infection. Furthermore, we demonstrated that polymeric 2F5 and 2G12 antibodies but not the corresponding IgGs could interfere with HIV-1 entry across a mucosal epithelial layer in vitro. Although polymeric 2F5 antibodies had only limited potential in the standard neutralization assay, the results from the mucosal assay suggest that 2F5 and 2G12 antibodies may have a high potential to prevent natural HIV-1 transmission in vivo.     

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.     

Passive immunization against oral AIDS virus transmission: An approach to prevent mother‐to‐infant HIV‐1 transmission?

To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.     

Vaccine with bacterium-like particles displaying HIV-1 gp120 trimer elicits specific mucosal responses and neutralizing antibodies in rhesus macaques

Preclinical studies have shown that the induction of secretory IgA (sIgA) in mucosa and neutralizing antibodies (NAbs) in sera is essential for designing vaccines that can effectively block the transmission of HIV-1. We previously showed that a vaccine consisting of bacterium-like particles (BLPs) displaying Protan-gp120AE-MTQ (PAM) could induce mucosal immune responses through intranasal (IN) immunization in mice and NAbs through intramuscular (IM) immunization in guinea pigs. Here, we evaluated the ability of this vaccine BLP-PAM to elicit HIV-1-specific mucosal and systemic immune responses through IN and IM immunization combination strategies in rhesus macaques. First, the morphology, antigenicity and epitope accessibility of the vaccine were analysed by transmission electron microscopy, bio-layer interferometry and ELISA. In BLP-PAM-immunized macaques, HIV-1-specific sIgA were rapidly induced through IN immunization in situ and distant mucosal sites, although the immune responses are relatively weak. Furthermore, the HIV-1-specific IgG and IgA antibody levels in mucosal secretions were enhanced and maintained, while production of serum NAbs against heterologous HIV-1 tier 1 and 2 pseudoviruses was elicited after IM boost. Additionally, situ mucosal responses and systemic T cell immune responses were improved by rAd2-gp120AE boost immunization via the IN and IM routes. These results suggested that BLP-based delivery in combination with the IN and IM immunization approach represents a potential vaccine strategy against HIV-1.     

Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.     

Detection of IgA-binding sites on human immunodeficiency virus type-1 envelope glycoproteins, Gp120 and Gp41

IgA has been supposed to play an important role in the prevention of HIV-1 infection. In this study, IgA-binding sites on gp120 and gp41 of HIV-1 envelope glycoproteins were analyzed using ELISA and overlapping synthetic peptides covering all of the gp120 and gp41 sites. IgA antibodies in plasma and saliva mainly bound to six and five sites on gp120 and gp41, respectively. Some of the IgA-binding sites differed from those of IgG-binding sites and the amount of IgA antibodies that bound to each site varied among samples. IgA antibodies in some plasma samples neutralized HIV-1 infection, and those IgA antibodies contained the antibodies which bound to the V3, C3 and ELDKWA sites. The results suggest that IgA antibodies which bind to certain sites on HIV-1 envelope glycoproteins may neutralize HIV-1 infection, presumably at mucosal sites where most IgA antibodies are produced. The induction of IgA antibodies that bind specific sites and neutralize HIV-1 infection at mucosal sites may be important in the development of a vaccine against HIV-1 infection.     

Mucosal model of genital immunization in male rhesus macaques with a recombinant simian immunodeficiency virus p27 antigen.

Human immunodeficiency virus (HIV) can be transmitted through infected seminal fluid or vaginal or rectal secretions during heterosexual or homosexual intercourse. To prevent mucosal transmission and spread to the regional lymph nodes, an effective vaccine may need to stimulate immune responses at the genitourinary mucosa. In this study, we have developed a mucosal model of genital immunization in male rhesus macaques, by topical urethral immunization with recombinant simian immunodeficiency virus p27gag, expressed as a hybrid Ty virus-like particle (Ty-VLP) and covalently linked to cholera toxin B subunit. This treatment was augmented by oral immunization with the same vaccine but with added killed cholera vibrios. Polymeric secretory immunoglobulin A (sIgA) and IgG antibodies to p27 were induced in urethral secretions, urine, and seminal fluid. This raises the possibility that the antibodies may function as a primary mucosal defense barrier against SIV (HIV) infection. The regional lymph nodes which constitute the genital-associated lymphoid tissue contained p27-specific CD4+ proliferative and helper T cells for antibody synthesis by B cells, which may function as a secondary immune barrier to infection. Blood and splenic lymphocytes also showed p27-sensitized CD4+ T cells and B cells in addition to serum IgG and IgA p27-specific antibodies; this constitutes a third level of immunity against dissemination of the virus. A comparison of genito-oral with recto-oral and intramuscular routes of immunization suggests that only genito-oral immunization elicits specific sIgA and IgG antibodies in the urine, urethra, and seminal fluid. Both genito-oral and recto-oral immunizations induced T-cell and B-cell immune responses in regional lymph nodes, with preferential IgA antibody synthesis. The mucosal route of immunization may prevent not only virus transmission through the genital mucosa but also dissemination and latency of the virus in the draining lymph nodes.     

An adenovirus-simian immunodeficiency virus env vaccine elicits humoral, cellular, and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge.

Six female rhesus macaques were immunized orally and intranasally at 0 weeks and intratracheally at 12 weeks with an adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus SIVsm env recombinant and at 24 and 36 weeks with native SIVmac251 gp120 in Syntex adjuvant. Four macaques received the Ad5hr vector and adjuvant alone; two additional controls were naive. In vivo replication of the Ad5hr wild-type and recombinant vectors occurred with detection of Ad5 DNA in stool samples and/or nasal secretions in all macaques and increases in Ad5 neutralizing antibody in 9 of 10 macaques following Ad administrations. SIV-specific neutralizing antibodies appeared after the second recombinant immunization and rose to titers > 10,000 following the second subunit boost. Immunoglobulin G (IgG) and IgA antibodies able to bind gp120 developed in nasal and rectal secretions, and SIV-specific IgGs were also observed in vaginal secretions and saliva. T-cell proliferative responses to SIV gp140 and T-helper epitopes were sporadically detected in all immunized macaques. Following vaginal challenge with SIVmac251, transient or persistent infection resulted in both immunized and control monkeys. The mean viral burden in persistently infected immunized macaques was significantly decreased in the primary infection period compared to that of control macaques. These results establish in vivo use of the Ad5hr vector, which overcomes the host range restriction of human Ads for rhesus macaques, thereby providing a new model for evaluation of Ad-based vaccines. In addition, they show that a vaccine regimen using the Ad5hr-SIV env recombinant and gp120 subunit induces strong humoral, cellular, and mucosal immunity in rhesus macaques. The reduced viral burden achieved solely with an env-based vaccine supports further development of Ad-based vaccines comprising additional viral components for immune therapy and AIDS vaccine development.     

Mucosal Immunization of Lactating Female Rhesus Monkeys with a Transmitted/Founder HIV-1 Envelope Induces Strong Env-Specific IgA Antibody Responses in Breast Milk

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.     

A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.     

Induction of mucosal and systemic neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) by oral immunization with bovine Papillomavirus-HIV-1 gp41 chimeric virus-like particles

Human immunodeficiency virus type 1 (HIV-1) envelope-specific neutralizing antibodies are generated late after initial infection, and the neutralizing antibody response is weak in the infected individuals. Administration of neutralizing antibodies such as 2F5 to HIV-1-infected individuals resulted in reductions in viral loads. Because HIV-1 is transmitted mainly via mucosa and because HIV-specific neutralizing antibodies reduce HIV-1 in infected individuals, a vaccine that can induce both mucosal and systemic HIV-1-specific neutralizing antibodies may be used to prevent and to treat HIV-1 infection. In this study, we made a bovine papillomavirus (BPV) L1-HIV-1 gp41 fusion protein in which ELDKWA of gp41 was inserted into the N terminus of BPV L1 (amino acids 130 to 136). Expression of the fusion protein in insect cells led to the assembly of chimeric virus-like particles (CVLPs). The CVLPs had sizes similar to those of BPV particles and were able to bind to the cell surface and penetrate the cell membrane. Oral immunization of mice with CVLPs induced gp41-specific serum immunoglobulin G (IgG) and intestinal secretory IgA. However, intramuscular immunization with the CVLPs resulted in similar amounts of gp41-specific IgG but low levels of secretory IgA. The antibodies specifically recognized the fixed HIV-1 gp41 on the cell surface. Importantly, the sera and fecal extracts from mice orally immunized with the CVLPs neutralized HIV-1(MN) in vitro. Thus, BPV-HIV-1 gp41 CVLPs may be used to prevent and to treat HIV-1 infection.     

Differential recognition of epitopes present on monomeric and oligomeric forms of gp160 glycoprotein of human immunodeficiency virus type 1 by human monoclonal antibodies.

The mechanism of infectivity neutralization of human immunodeficiency virus type 1 (HIV-1) by Ig is poorly understood. Three human monoclonal antibodies (mAbs 1b12, 2G12 and 2F5) that are able to neutralize primary isolates of HIV-1 in vitro have been shown to act synergistically. In the present study this synergy was analyzed by measuring the epitope accessibility and binding kinetics for these three mAbs with respect to monomeric and oligomeric env protein gp160 IIIB using surface plasmon resonance. The results indicate that oligomerization of gp160 affects the accessibility of some of the epitopes recognized by the mAbs and provide some insight into the mechanism of synergy between different anti-(HIV-1) mAbs.     

Systemic immunization with an ALVAC-HIV-1/protein boost vaccine strategy protects rhesus macaques from CD4+ T-cell loss and reduces both systemic and mucosal simian-human immunodeficiency virus SHIVKU2 RNA levels

Transmission of human immunodeficiency virus type 1 (HIV-1) occurs primarily via the mucosal route, suggesting that HIV-1 vaccines may need to elicit mucosal immune responses. Here, we investigated the immunogenicity and relative efficacy of systemic immunization with two human ALVAC-HIV-1 recombinant vaccines expressing Gag, Pol, and gp120 (vCP250) or Gag, Pol, and gp160 (vCP1420) in a prime-boost protocol with their homologous vaccine native Env proteins. The relative efficacy was measured against a high-dose mucosal exposure to the pathogenic neutralization-resistant variant SHIV(KU2) (simian-human immunodeficiency virus). Systemic immunization with both vaccine regimens decreased viral load levels not only in blood but unexpectedly also in mucosal sites and protected macaques from peripheral CD4+ T-cell loss. This protective effect was stronger when the gp120 antigen was included in the vaccine. Inclusion of recombinant Tat protein in the boosting phase along with the Env protein did not contribute further to the preservation of CD4+ T cells. Thus, systemic immunization with ALVAC-HIV-1 vaccine candidates elicits anti-HIV-1 immune responses able to contain virus replication also at mucosal sites in macaques.     

Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.     

Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection

Two human monoclonal antibodies (MAbs) (2F5 and 4E10) against the human immunodeficiency virus type 1 (HIV-1) envelope g41 cluster II membrane proximal external region (MPER) broadly neutralize HIV-1 primary isolates. However, these antibody specificities are rare, are not induced by Env immunization or HIV-1 infection, and are polyspecific and also react with lipids such as cardiolipin or phosphatidylserine. To probe MPER anti-gp41 antibodies that are produced in HIV-1 infection, we have made two novel murine MAbs, 5A9 and 13H11, against HIV-1 gp41 envelope that partially cross-blocked 2F5 MAb binding to Env but did not neutralize HIV-1 primary isolates or bind host lipids. Competitive inhibition assays using labeled 13H11 MAb and HIV-1-positive patient plasma samples demonstrated that cluster II 13H11-blocking plasma antibodies were made in 83% of chronically HIV-1 infected patients and were acquired between 5 to 10 weeks after acute HIV-1 infection. Both the mouse 13H11 MAb and the three prototypic cluster II human MAbs (98-6, 126-6, and 167-D) blocked 2F5 binding to gp41 epitopes to variable degrees; the combination of 98-6 and 13H11 completely blocked 2F5 binding. These data provide support for the hypothesis that in some patients, B cells make nonneutralizing cluster II antibodies that may mask or otherwise down-modulate B-cell responses to immunogenic regions of gp41 that could be recognized by B cells capable of producing antibodies like 2F5.     

Intranasal vaccination using interleukin-12 and cholera toxin subunit B as adjuvants to enhance mucosal and systemic immunity to human immunodeficiency virus type 1 glycoproteins

We have investigated the induction of protective mucosal immunity to human immunodeficiency virus type 1 (HIV-1) isolate 89.6 by intranasal (i.n.) immunization of mice with gp120 and gp140 together with interleukin-12 (IL-12) and cholera toxin subunit B (CTB) as adjuvants. It was found that both IL-12 and CTB were required to elicit mucosal antibody responses and that i.n. immunization resulted in increased total, immunoglobulin G1 (IgG1), and IgG2a anti-HIV-1 antibody levels in serum; increased total, IgG1, IgG2a, and IgA antibody expression in bronchoalveolar lavage fluids; and increased IgA antibody levels in vaginal washes. Levels of anti-HIV-1 antibodies in both sera and secretions were higher in groups immunized with gp140 than in those immunized with gp120. However, only gp120-specific mucosal antibodies demonstrated neutralizing activity against HIV-1 89.6. Taken together, the results show that IL-12 and CTB act synergistically to enhance both systemic and local mucosal antibody responses to HIV-1 glycoproteins and that even though gp140 induces higher antibody titers than gp120, only gp120-specific mucosal antibodies interfere with virus infectivity.     

Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG.

We have tested three human monoclonal antibodies (MAbs) IgG1b12, 2G12, and 2F5) to the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1), and a tetrameric CD4-IgG molecule (CD4-IgG2), for the ability to neutralize primary HIV-1 isolates from the genetic clades A through F and from group O. Each of the reagents broadly and potently neutralized B-clade isolates. The 2F5 MAb and the CD4-IgG2 molecule also neutralized strains from outside the B clade, with the same breadth and potency that they showed against B-clade strains. The other two MAbs were able to neutralize a significant proportion of strains from outside the B clade, although there was a reduction in their efficacy compared with their activity against B-clade isolates. Neutralization of isolates by 2F5 correlated with their possession of the LDKW motif in a segment of gp41 near the membrane-spanning domain. The other two MAbs and CD4-IgG2 recognize discontinuous binding sites on gp120, and so no comparison between genetic sequence and virus neutralization was possible. Our data show that a vaccine based on the induction of humoral immunity that is broadly active across the genetic clades is not impossible if immunogens that express the epitopes for MAbs such as 2F5, 2G12, and IgG1b12 in immunogenic configurations can be created. Furthermore, if the three MAbs and CD4-IgG2 produce clinical benefit in immunotherapeutic trials in the United States or Europe, they may also do so elsewhere in the world.     

Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge

Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.