Judith A. Burger

Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes

The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs.

Rapid Escape from Preserved Cross-Reactive Neutralizing Humoral Immunity without Loss of Viral Fitness in HIV-1-Infected Progressors and Long-Term Nonprogressors

ABSTRACT A substantial proportion of human immunodeficiency virus type 1 (HIV-1)-infected individuals has cross-reactive neutralizing activity in serum, with a similar prevalence in progressors and long-term nonprogressors (LTNP). We studied whether disease progression in the face of cross-reactive neutralizing serum activity is due to fading neutralizing humoral immunity over time or to viral escape. In three LTNP and three progressors, high-titer cross-reactive HIV-1-specific neutralizing activity in serum against a multiclade pseudovirus panel was preserved during the entire clinical course of infection, even after AIDS diagnosis in progressors. However, while early HIV-1 variants from all six individuals could be neutralized by autologous serum, the autologous neutralizing activity declined during chronic infection. This could be attributed to viral escape and the apparent inability of the host to elicit neutralizing antibodies to the newly emerging viral escape variants. Escape from autologous neutralizing activity was not associated with a reduction in the viral replication rate in vitro. Escape from autologous serum with cross-reactive neutralizing activity coincided with an increase in the length of the variable loops and in the number of potential N-linked glycosylation sites in the viral envelope. Positive selection pressure was observed in the variable regions in envelope, suggesting that, at least in these individuals, these regions are targeted by humoral immunity with cross-reactive potential. Our results may imply that the ability of HIV-1 to rapidly escape cross-reactive autologous neutralizing antibody responses without the loss of viral fitness is the underlying explanation for the absent effect of potent cross-reactive neutralizing humoral immunity on the clinical course of infection.

Longer V1V2 Region with Increased Number of Potential N-Linked Glycosylation Sites in the HIV-1 Envelope Glycoprotein Protects against HIV-Specific Neutralizing Antibodies

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) has the ability to adapt to the host environment by escaping from host immune responses. We previously observed that escape from humoral immunity, both at the individual and at a population level, coincided with longer variable loops and an increased number of potential N-linked glycosylation sites (PNGS) in the viral envelope glycoprotein (Env) and, in particular, in variable regions 1 and 2 (V1V2). Here, we provide several lines of evidence for the role of V1V2 in the resistance of HIV-1 to neutralizing antibodies. First, we determined that the increasing neutralization resistance of a reference panel of tier-categorized neutralization-sensitive and -resistant HIV-1 variants coincided with a longer V1V2 loop containing more PNGS. Second, an exchange of the different variable regions of Env from a neutralization-sensitive HIV-1 variant into a neutralization-resistant escape variant from the same individual revealed that the V1V2 loop is a strong determinant for sensitivity to autologous-serum neutralization. Third, exchange of the V1V2 loop of neutralization-sensitive HIV-1 variants from historical seroconverters with the V1V2 loop of neutralization-resistant HIV-1 variants from contemporary seroconverters decreased the neutralization sensitivity to CD4-binding site-directed antibodies. Overall, we demonstrate that an increase in the length of the V1V2 loop and/or the number of PNGS in that same region of the HIV-1 envelope glycoprotein is directly involved in the protection of HIV-1 against HIV-specific neutralizing antibodies, possibly by shielding underlying epitopes in the envelope glycoprotein from antibody recognition.

Longitudinal analysis of early HIV-1 specific neutralizing activity in an elite neutralizer and in five patients who developed cross-reactive neutralizing activity

ABSTRACT We previously established that at 3 years postseroconversion, ∼30% of HIV-infected individuals have cross-reactive neutralizing activity (CrNA) in their sera. Here we studied the kinetics with which CrNA develops and how these relate to the development of autologous neutralizing activity as well as viral escape and diversification. For this purpose, sera from five individuals with CrNA and one elite neutralizer that were obtained at three monthly intervals in the first year after seroconversion and at multiple intervals over the disease course were tested for neutralizing activity against an established multiclade panel of six viruses. The same serum samples, as well as sera from three individuals who lacked CrNA, were tested for their neutralizing activities against autologous clonal HIV-1 variants from multiple time points covering the disease course from seroconversion onward. The elite neutralizer already had CrNA at 9.8 months postseroconversion, in contrast with the findings for the other five patients, in whom CrNA was first detected at 20 to 35 months postseroconversion and peaked around 35 months postseroconversion. In all patients, CrNA coincided with neutralizing activity against autologous viruses that were isolated <12 months postseroconversion, while viruses from later time points had already escaped autologous neutralizing activity. Also, the peak in gp160 sequence diversity coincided with the peak of CrNA titers. Individuals who lacked CrNA had lower peak autologous neutralizing titers, viral escape, and sequence diversity than individuals with CrNA. A better understanding of the underlying factors that determine the presence of CrNA or even an elite neutralizer phenotype may aid in the design of an HIV-1 vaccine.

Activity of Broadly Neutralizing Antibodies, Including PG9, PG16, and VRC01, against Recently Transmitted Subtype B HIV-1 Variants from Early and Late in the Epidemic

ABSTRACT For the development of a neutralizing antibody-based human immunodeficiency virus type 1 (HIV-1) vaccine, it is important to characterize which antibody specificities are most effective against currently circulating HIV-1 variants. We recently reported that HIV-1 has become more resistant to antibody neutralization over the course of the epidemic, and we here explore whether this increased neutralization resistance is also observed for the newly identified broadly neutralizing antibodies (BrNAbs) PG9, PG16, and VRC01. Furthermore, we performed a comprehensive analysis of the neutralizing sensitivity of currently circulating recently transmitted subtype B viruses to the currently most known BrNAbs. Virus variants isolated less than 6 months after seroconversion from individuals who seroconverted between 2003 and 2006 (n = 21) were significantly more resistant to neutralization by VRC01 than viruses from individuals who seroconverted between 1985 and 1989 (n = 14). In addition, viruses from contemporary seroconverters tended to be more resistant to neutralization by PG16, which coincided with the presence of more mutations at positions in the viral envelope that may potentially influence neutralization by this antibody. Despite this increased neutralization resistance, all recently transmitted viruses from contemporary seroconverters were sensitive to at least one BrNAb at concentrations of ≤5 μg/ml, with PG9, PG16, and VRC01 showing the greatest breadth of neutralization at lower concentrations. These results suggest that a vaccine capable of eliciting multiple BrNAb specificities will be necessary for protection of the population against HIV-1 infection.

Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies

The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design.

Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicity.

BackgroundPresenting vaccine antigens in particulate form can improve their immunogenicity by enhancing B cell activation.FindingsWe describe ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664). Trimer-bearing nanoparticles were significantly more immunogenic than trimers in both mice and rabbits. Furthermore, rabbits immunized with the trimer-bearing nanoparticles induced significantly higher neutralizing antibody responses against most tier 1A viruses, and higher responses (but not significantly), to several tier 1B viruses and the autologous tier 2 virus than when the same trimers were delivered as soluble proteins.ConclusionsThis or other nanoparticle designs may be practical ways to improve the immunogenicity of envelope glycoprotein trimers.

Genome-Wide Association Scan in HIV-1-Infected Individuals Identifying Variants Influencing Disease Course

Background AIDS develops typically after 7–11 years of untreated HIV-1 infection, with extremes of very rapid disease progression (<2 years) and long-term non-progression (>15 years). To reveal additional host genetic factors that may impact on the clinical course of HIV-1 infection, we designed a genome-wide association study (GWAS) in 404 participants of the Amsterdam Cohort Studies on HIV-1 infection and AIDS. Methods The association of SNP genotypes with the clinical course of HIV-1 infection was tested in Cox regression survival analyses using AIDS-diagnosis and AIDS-related death as endpoints. Results Multiple, not previously identified SNPs, were identified to be strongly associated with disease progression after HIV-1 infection, albeit not genome-wide significant. However, three independent SNPs in the top ten associations between SNP genotypes and time between seroconversion and AIDS-diagnosis, and one from the top ten associations between SNP genotypes and time between seroconversion and AIDS-related death, had P-values smaller than 0.05 in the French Genomics of Resistance to Immunodeficiency Virus cohort on disease progression. Conclusions Our study emphasizes that the use of different phenotypes in GWAS may be useful to unravel the full spectrum of host genetic factors that may be associated with the clinical course of HIV-1 infection.

Improving the Immunogenicity of Native-like HIV-1 Envelope Trimers by Hyperstabilization

Summary The production of native-like recombinant versions of the HIV-1 envelope glycoprotein (Env) trimer requires overcoming the natural flexibility and instability of the complex. The engineered BG505 SOSIP.664 trimer mimics the structure and antigenicity of native Env. Here, we describe how the introduction of new disulfide bonds between the glycoprotein (gp)120 and gp41 subunits of SOSIP trimers of the BG505 and other genotypes improves their stability and antigenicity, reduces their conformational flexibility, and helps maintain them in the unliganded conformation. The resulting next-generation SOSIP.v5 trimers induce strong autologous tier-2 neutralizing antibody (NAb) responses in rabbits. In addition, the BG505 SOSIP.v6 trimers induced weak heterologous NAb responses against a subset of tier-2 viruses that were not elicited by the prototype BG505 SOSIP.664. These stabilization methods can be applied to trimers from multiple genotypes as components of multivalent vaccines aimed at inducing broadly NAbs (bNAbs).

Absence of HIV-1 Superinfection 1 Year after Infection between 1985 and 1997 Coincides with a Reduction in Sexual Risk Behavior in the Seroincident Amsterdam Cohort of Homosexual Men

BACKGROUND Incidence rates of human immunodeficiency virus type 1 (HIV-1) superinfection differ among cohorts and, as yet, only 2 cohorts of homosexual men have been screened. Here, we investigated the incidence of HIV-1 superinfection during the first year after infection among homosexual participants in the Amsterdam Cohort Studies on HIV infection and AIDS who seroconverted between 1985 and 1997. METHODS We analyzed env C2-C4 diversity in the serum of therapy-naive participants, using a heteroduplex mobility assay; heteroduplexes were considered to be indicators of potential dual infections, in which case env C2-C4 polymerase chain reaction (PCR) products were cloned and sequenced. Sequences were subjected to phylogenetic analysis. Data on the sexual behavior of participants were collected from 1 year before seroconversion until the end of the investigated period. RESULTS For 89 seroconverters with a detectable viral load (>1000 copies/mL), env PCR products were generated from serum samples obtained at seroconversion and 1 year later. Heteroduplexes were observed in 68 of the 89 patients; among these 68 patients, a median of 9 molecular clones per time point was sequenced. Phylogenetic analysis did not reveal evidence for superinfection; 1 patient was HIV-1 coinfected. Shortly after diagnosis of HIV infection, the number of sex partners decreased, the frequency of anal intercourse declined, and condom use increased. CONCLUSIONS The incidence of HIV-1 superinfection soon after seroconversion in this cohort is low. Risk reduction shortly after HIV-1 diagnosis early during the HIV-1 epidemic in the Netherlands may have contributed to the absence of HIV-1 superinfection observed in this study.