Scientific Publications

2G12 is a broadly neutralizing anti-HIV-1 monoclonal human IgG1 antibody reactive with a high-mannose glycan cluster on the surface of glycoprotein gp120. A key feature of this very highly mutated antibody is domain exchange of the heavy-chain variable region (V(H)) with the V(H) of the adjacent Fab of the same immunoglobulin, which assembles a multivalent binding interface composed of two primary binding sites in close proximity. A non-germ line-encoded proline in the elbow between V(H) and C(H)1 and an extensive network of hydrophobic interactions in the V(H)/V(H)' interface have been proposed to be crucial for domain exchange. To investigate the origins of domain exchange, a germ line version of 2G12 that behaves as a conventional antibody was engineered. Substitution of 5 to 7 residues for those of the wild type produced a significant fraction of domain-exchanged molecules, with no evidence of equilibrium between domain-exchanged and conventional forms. Two substitutions not previously implicated, A(H14) and E(H75), are the most crucial for domain exchange, together with I(H19) at the V(H)/V(H)' interface and P(H113) in the elbow region. Structural modeling gave clues as to why these residues are essential for domain exchange. The demonstration that domain exchange can be initiated by a small number of substitutions in a germ line antibody suggests that the evolution of a domain-exchanged antibody response in vivo may be more readily achieved than considered to date.

Since HIV-1 was identified, development of a preventive vaccine has been a major goal. Significant progress toward that goal has been made by 2010. In macaques, a vigorous T effector cell response has protected some animals from disease caused by simian immunodeficiency virus (SIV). Broadly, neutralizing human anti-HIV antibodies have been isolated and their structures, and targets are rapidly being elucidated. For the first time an AIDS vaccine has shown modest protective efficacy in a human clinical trial. To reach the final goal, there is a need for a coordinated global effort, including a range of approaches including novel high-throughput screening techniques, X-ray crystallography, and monoclonal antibody isolation, analysis of T cell responses and their impact on disease progression, human epidemiology, as well as targeted studies in nonhuman primates. African research teams as well as cohorts of healthy volunteers and HIV-infected individuals have contributed to HIV vaccine research and development in many important ways. It is essential that this work continue to speed the development and deployment of a vaccine suitable for African populations.

The broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibody 2G12 targets the high-mannose cluster on the glycan shield of HIV-1. 2G12 has a unique V(H) domain-exchanged structure, with a multivalent binding surface that includes two primary glycan binding sites. The high-mannose cluster is an attractive target for HIV-1 vaccine design, but so far, no carbohydrate immunogen has elicited 2G12-like antibodies. Important questions remain as to how this domain exchange arose in 2G12 and how this unusual event conferred unexpected reactivity against the glycan shield of HIV-1. In order to address these questions, we generated a nondomain-exchanged variant of 2G12 to produce a conventional Y/T-shaped antibody through a single amino acid substitution (2G12 I19R) and showed that, as for the 2G12 wild type (2G12 WT), this antibody is able to recognize the same Manα1,2Man motif on recombinant gp120, Candida albicans, and synthetic glycoconjugates. However, the nondomain-exchanged variant of 2G12 is unable to bind the cluster of mannose moieties on the surface of HIV-1. Crystallographic analysis of 2G12 I19R in complex with Manα1,2Man revealed an adaptable hinge between V(H) and C(H)1 that enables the V(H) and V(L) domains to assemble in such a way that the configuration of the primary binding site and its interaction with disaccharide are remarkably similar in the nondomain-exchanged and domain-exchanged forms. Together with data that suggest that very few substitutions are required for domain exchange, the results suggest potential mechanisms for the evolution of domain-exchanged antibodies and immunization strategies for eliciting such antibodies.

The HIV-1-specific antibodies PG9 and PG16 show marked cross-isolate neutralization breadth and potency. Antibody neutralization has been shown to be dependent on the presence of N-linked glycosylation at position 160 in gp120. We show here that (i) the loss of several key glycosylation sites in the V1, V2, and V3 loops; (ii) the generation of pseudoviruses in the presence of various glycosidase inhibitors; and (iii) the growth of pseudoviruses in a mutant cell line (GnT1(-/-)) that alters envelope glycosylation patterns all have significant effects on the sensitivity of virus to neutralization by PG9 and PG16. However, the interaction of antibody is not inhibited by sugar monosaccharides corresponding to those found in glycans on the HIV surface. We show that some of the glycosylation effects described are isolate dependent and others are universal and can be used as diagnostic for the presence of PG9 and PG16-like antibodies in the sera of HIV-1-infected patients. The results suggest that PG9 and PG16 recognize a conformational epitope that is dependent on glycosylation at specific variable loop N-linked sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies.

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.

We conducted a double-blind, randomized, placebo-controlled Phase I study of a recombinant replication-defective adenovirus type 5 (rAd5) vector expressing HIV-1 Gag and Pol from subtype B and Env from subtypes A, B and C, given alone or as boost following a DNA plasmid vaccine expressing the same HIV-1 proteins plus Nef, in 114 healthy HIV-uninfected African adults.

The symposium 'HIV/AIDS: Vaccines and Alternate Strategies for Treatment and Prevention' brought together HIV vaccine researchers to discuss the latest developments in the field. From basic discoveries in virus diversity and mechanisms of neutralization by antibodies to nonhuman primate research and clinical trials of vaccine candidates in volunteers, scientists are making great strides in understanding the mechanisms that may protect against HIV and pathways to achieve this protection through vaccination.

The virus-encoded envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) typically contain 26 to 30 sites for N-linked carbohydrate attachment. N-linked carbohydrate can be of three major types: high mannose, complex, or hybrid. The lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA), which specifically bind high-mannose carbohydrate, were found to potently inhibit the replication of a pathogenic cloned SIV from rhesus macaques, SIVmac239. Passage of SIVmac239 in the presence of escalating concentrations of GNA and HHA yielded a lectin-resistant virus population that uniformly eliminated three sites (of 26 total) for N-linked carbohydrate attachment (Asn-X-Ser or Asn-X-Thr) in the envelope protein. Two of these sites were in the gp120 surface subunit of the envelope protein (Asn244 and Asn460), and one site was in the envelope gp41 transmembrane protein (Asn625). Maximal resistance to GNA and HHA in a spreading infection was conferred to cloned variants that lacked all three sites in combination. Variant SIV gp120s exhibited dramatically decreased capacity for binding GNA compared to SIVmac239 gp120 in an enzyme-linked immunosorbent assay (ELISA). Purified gp120s from six independent HIV type 1 (HIV-1) isolates and two SIV isolates from chimpanzees (SIVcpz) consistently bound GNA in ELISA at 3- to 10-fold-higher levels than gp120s from five SIV isolates from rhesus macaques or sooty mangabeys (SIVmac/sm) and four HIV-2 isolates. Thus, our data indicate that characteristic high-mannose carbohydrate contents have been retained in the cross-species transmission lineages for SIVcpz-HIV-1 (high), SIVsm-SIVmac (low), and SIVsm-HIV-2 (low).

An effective human immunodeficiency virus (HIV) vaccine will likely need to reduce mucosal transmission and, if infection occurs, control virus replication. To determine whether our best simian immunodeficiency virus (SIV) vaccine can achieve these lofty goals, we vaccinated eight Indian rhesus macaques with SIVmac239Delta nef and challenged them intrarectally (i.r.) with repeated low doses of the pathogenic heterologous swarm isolate SIVsmE660. We detected a significant reduction in acquisition of SIVsmE660 in comparison to that for naïve controls (log rank test; P = 0.023). After 10 mucosal challenges, we detected replication of the challenge strain in only five of the eight vaccinated animals. In contrast, seven of the eight control animals became infected with SIVsmE660 after these 10 challenges. Additionally, the SIVsmE660-infected vaccinated animals controlled peak acute virus replication significantly better than did the naïve controls (Mann-Whitney U test; P = 0.038). Four of the five SIVsmE660 vaccinees rapidly brought virus replication under control by week 4 postinfection. Unfortunately, two of these four vaccinated animals lost control of virus replication during the chronic phase of infection. Bulk sequence analysis of the circulating viruses in these animals indicated that recombination had occurred between the vaccine and challenge strains and likely contributed to the increased virus replication in these animals. Overall, our results suggest that a well-designed HIV vaccine might both reduce the rate of acquisition and control viral replication.

Little is known about how the information presented in the informed consent process influences study outcomes among participants. This study examines the influence of informed consent content on reported baseline contraceptive knowledge and concerns among two groups of HIV-serodiscordant and seroconcordant HIV-positive couples enrolled in research projects at an HIV research center in Lusaka, Zambia. We found significant differences in the reporting of contraceptive knowledge and concerns between couples viewing consent materials that included detailed information about contraception and those viewing consent materials that lacked the detailed information. We conclude that the design of informed consent materials should strike a balance between ensuring that participants give truly informed consent and educating participants in ways that do not compromise the assessment of the impact of behavioral interventions.

Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoproteins (Envs) have proven difficult to elicit by immunization. Therefore, to identify effective Env neutralization targets, efforts are underway to define the specificities of bNAbs in chronically infected individuals. For a prophylactic vaccine, it is equally important to define the immunogenic properties of the heavily glycosylated Env in healthy primates devoid of confounding HIV-induced pathogenic factors. We used rhesus macaques to investigate the magnitude and kinetics of B cell responses stimulated by Env trimers in adjuvant. Robust Env-specific memory B cell responses and high titers of circulating antibodies developed after trimer inoculation. Subsequent immunizations resulted in significant expansion of Env-specific IgG-producing plasma cell populations and circulating Abs that displayed increasing avidity and neutralization capacity. The neutralizing activity elicited with the regimen used was, in most aspects, superior to that elicited by a regimen based on monomeric Env immunization in humans. Despite the potency and breadth of the trimer-elicited response, protection against heterologous rectal simian-HIV (SHIV) challenge was modest, illustrating the challenge of eliciting sufficient titers of cross-reactive protective NAbs in mucosal sites. These data provide important information for the design and evaluation of vaccines aimed at stimulating protective HIV-1 immune responses in humans.

The outer domain (OD) of the HIV-1 envelope glycoprotein gp120 is an important target for vaccine design as it contains a number of conserved epitopes, including a large fraction of the CD4 binding site. Attempts to design OD-based immunogens in the past have met with little success. We report the design and characterization of an Escherichia coli-expressed OD-based immunogen (OD(EC)), based on the sequence of the HxBc2 strain. The OD(EC)-designed immunogen lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gp120. Biophysical studies showed that OD(EC) is folded and protease-resistant, whereas OD(EC) lacking the designed mutations is highly aggregation-prone. In contrast to previously characterized OD constructs, OD(EC) bound CD4 and the broadly neutralizing antibody b12 but not the non-neutralizing antibodies b6 and F105. Upon immunization in rabbits, OD(EC) was highly immunogenic, and the sera showed measurable neutralization for four subtype B and one subtype C virus including two b12-resistant viruses. In contrast, sera from rabbits immunized with gp120 did not neutralize any of the viruses. OD(EC) is the first example of a gp120 fragment-based immunogen that yields significant neutralizing antibodies.