Leonidas Stamatatos

Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies

ABSTRACT Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.

Factors Associated with the Development of Cross-Reactive Neutralizing Antibodies during Human Immunodeficiency Virus Type 1 Infection

ABSTRACT The characterization of the cross-reactive, or heterologous, neutralizing antibody responses developed during human immunodeficiency virus type 1 (HIV-1) infection and the identification of factors associated with their generation are relevant to the development of an HIV vaccine. We report that in healthy HIV-positive, antiretroviral-naïve subjects, the breadth of plasma heterologous neutralizing antibody responses correlates with the time since infection, plasma viremia levels, and the binding avidity of anti-Env antibodies. Anti-CD4-binding site antibodies are responsible for the exceptionally broad cross-neutralizing antibody responses recorded only in rare plasma samples. However, in most cases examined, antibodies to the variable regions and to the CD4-binding site of Env modestly contributed in defining the overall breadth of these responses. Plasmas with broad cross-neutralizing antibody responses were identified that targeted the gp120 subunit, but their precise epitopes mapped outside the variable regions and the CD4-binding site. Finally, although several plasmas were identified with cross-neutralizing antibody responses that were not directed against gp120, only one plasma with a moderate breadth of heterologous neutralizing antibody responses contained cross-reactive neutralizing antibodies against the 4E10 epitope, which is within the gp41 transmembrane subunit. Overall, our study indicates that more than one pathway leads to the development of broad cross-reactive neutralizing antibodies during HIV infection and that the virus continuously escapes their action.

Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine?

Most existing viral vaccines generate antibodies that either block initial infection or help eradicate the virus before it can cause disease. For HIV-1, obstacles to eliciting protective neutralizing antibodies (NAbs) have often seemed insurmountable. The target of HIV-specific NAbs, the viral envelope glycoprotein (Env), is highly variable in amino acid sequence and glycosylation pattern. Conserved elements of HIV-1 Env seem to be poorly immunogenic, and previous attempts to generate broadly reactive NAbs by vaccination have proven ineffective. However, recent studies show that antibodies in the sera of some HIV-1–infected individuals can neutralize diverse HIV-1 isolates. Detailed analyses of these sera provide new insights into the viral epitopes targeted by broadly reactive NAbs. The findings discussed here suggest that the natural NAb response to HIV-1 can inform future vaccine design. A concerted effort of structure-based vaccine design will help guide the development of improved antibody-based vaccines for HIV-1.

Rational HIV Immunogen Design to Target Specific Germline B Cell Receptors

Building Better Vaccines In the past few years, several highly potent, broadly neutralizing antibodies (bNAbs) specific for the gp120 envelope protein of HIV-1 have been discovered. The goal of this work is to use this information to inform the design of vaccines that are able to induce such antibodies (see the Perspective by Crowe). However, because of extensive somatic hypermutation, the epitope bound by these antibodies often does not bind to the germline sequence. Jardine et al. (p. 711, published online 28 March; see the cover) used computational analysis and in vitro screening to design an immunogen that could bind to VRC01-class bNAbs and to their germline precursors. Georgiev et al. (p. 751) took advantage of the fact that only four sites on the HIV viral envelope protein seem to bind bNAbs, and sera that contain particular bNAbs show characteristic patterns of neutralization. An algorithm was developed that could successfully delineate the neutralization specificity of antibodies present in polyclonal sera from HIV-infected patients. Structural knowledge of broadly neutralizing antibodies against HIV-1 guides the design of an immunogen to elicit them. Vaccine development to induce broadly neutralizing antibodies (bNAbs) against HIV-1 is a global health priority. Potent VRC01-class bNAbs against the CD4 binding site of HIV gp120 have been isolated from HIV-1–infected individuals; however, such bNAbs have not been induced by vaccination. Wild-type gp120 proteins lack detectable affinity for predicted germline precursors of VRC01-class bNAbs, making them poor immunogens to prime a VRC01-class response. We employed computation-guided, in vitro screening to engineer a germline-targeting gp120 outer domain immunogen that binds to multiple VRC01-class bNAbs and germline precursors, and elucidated germline binding crystallographically. When multimerized on nanoparticles, this immunogen (eOD-GT6) activates germline and mature VRC01-class B cells. Thus, eOD-GT6 nanoparticles have promise as a vaccine prime. In principle, germline-targeting strategies could be applied to other epitopes and pathogens.

The Ability of an Oligomeric Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Antigen To Elicit Neutralizing Antibodies against Primary HIV-1 Isolates Is Improved following Partial Deletion of the Second Hypervariable Region

ABSTRACT Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162ΔV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840–7845, 1998). This observation led us to propose that the modified, SF162ΔV2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162ΔV2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162ΔV2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.

Characteristics of the Earliest Cross-Neutralizing Antibody Response to HIV-1

Recent cross-sectional analyses of HIV-1+ plasmas have indicated that broadly cross-reactive neutralizing antibody responses are developed by 10%–30% of HIV-1+ subjects. The timing of the initial development of such anti-viral responses is unknown. It is also unknown whether the emergence of these responses coincides with the appearance of antibody specificities to a single or multiple regions of the viral envelope glycoprotein (Env). Here we analyzed the cross-neutralizing antibody responses in longitudinal plasmas collected soon after and up to seven years after HIV-1 infection. We find that anti-HIV-1 cross-neutralizing antibody responses first become evident on average at 2.5 years and, in rare cases, as early as 1 year following infection. If cross-neutralizing antibody responses do not develop during the first 2–3 years of infection, they most likely will not do so subsequently. Our results indicate a potential link between the development of cross-neutralizing antibody responses and specific activation markers on T cells, and with plasma viremia levels. The earliest cross-neutralizing antibody response targets a limited number of Env regions, primarily the CD4-binding site and epitopes that are not present on monomeric Env, but on the virion-associated trimeric Env form. In contrast, the neutralizing activities of plasmas from subjects that did not develop cross-neutralizing antibody responses target epitopes on monomeric gp120 other than the CD4-BS. Our study provides information that is not only relevant to better understanding the interaction of the human immune system with HIV but may guide the development of effective immunization protocols. Since antibodies to complex epitopes that are present on the virion-associated envelope spike appear to be key components of earliest cross-neutralizing activities of HIV-1+ plasmas, then emphasis should be made to elicit similar antibodies by vaccination.

Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies.

Eliminating key glycosylation sites on HIV envelope (Env) restores binding of the germline versions of known broadly neutralizing anti-Env antibodies.

Purification, Characterization, and Immunogenicity of a Soluble Trimeric Envelope Protein Containing a Partial Deletion of the V2 Loop Derived from SF162, an R5-Tropic Human Immunodeficiency Virus Type 1 Isolate

ABSTRACT The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162ΔV2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIVSF162P4 virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (Rh) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected Mw of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162ΔV2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162ΔV2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162ΔV2 Env immunogen in a successful HIV vaccine.

V2 Loop Glycosylation of the Human Immunodeficiency Virus Type 1 SF162 Envelope Facilitates Interaction of This Protein with CD4 and CCR5 Receptors and Protects the Virus from Neutralization by Anti-V3 Loop and Anti-CD4 Binding Site Antibodies

ABSTRACT We examined the role of asparagine-linked glycosylation of the V2 loop of the human immunodeficiency virus (HIV) SF162 envelope on viral replication potential and neutralization susceptibility. We report that the asparagines located at the amino- and carboxy-terminal sites (at positions 154 and 195, respectively), as well as within the V2 loop of the SF162 envelope (at position 186), are glycosylated during in vitro replication of this virus in human peripheral blood mononuclear cells. Our studies indicate that glycosylation of the V2 loop, in particular at its base, facilitates the interaction of the HIV envelope with the CD4 and CCR5 receptor molecules present on the surface of target cells and affects viral replication kinetics in a cell type-dependent manner. In cells expressing high numbers of receptor molecules on their surfaces, the SF162-derived V2 loop-deglycosylated mutant viruses replicate as efficiently as the parental SF162 virus, while in cells expressing small numbers of receptor molecules, the mutant viruses replicate with markedly reduced efficiency. In addition to expanding the viral tropism, V2 loop glycosylation at the three sites examined prevents neutralization by anti-CD4 binding site antibodies. In contrast, glycosylation at the amino- and carboxy-terminal sites of the V2 loop but not within the loop itself offers protection against anti-V3 loop antibodies. Thus, the epitopes masked by the sugar molecules present on the three glycosylation sites examined are not identical but overlap.

Computational Design of Epitope-Scaffolds Allows Induction of Antibodies Specific for a Poorly Immunogenic HIV Vaccine Epitope.

Broadly cross-reactive monoclonal antibodies define epitopes for vaccine development against HIV and other highly mutable viruses. Crystal structures are available for several such antibody-epitope complexes, but methods are needed to translate that structural information into immunogens that re-elicit similar antibodies. We describe a general computational method to design epitope-scaffolds in which contiguous structural epitopes are transplanted to scaffold proteins for conformational stabilization and immune presentation. Epitope-scaffolds designed for the poorly immunogenic but conserved HIV epitope 4E10 exhibited high epitope structural mimicry, bound with higher affinities to monoclonal antibody (mAb) 4E10 than the cognate peptide, and inhibited HIV neutralization by HIV+ sera. Rabbit immunization with an epitope-scaffold induced antibodies with structural specificity highly similar to mAb 4E10, an important advance toward elicitation of neutralizing activity. The results demonstrate that computationally designed epitope-scaffolds are valuable as structure-specific serological reagents and as immunogens to elicit antibodies with predetermined structural specificity.