Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC₅₀) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.     

HIV-1 envelope subregion length variation during disease progression

The V3 loop of the HIV-1 Env protein is the primary determinant of viral coreceptor usage, whereas the V1V2 loop region is thought to influence coreceptor binding and participate in shielding of neutralization-sensitive regions of the Env glycoprotein gp120 from antibody responses. The functional properties and antigenicity of V1V2 are influenced by changes in amino acid sequence, sequence length and patterns of N-linked glycosylation. However, how these polymorphisms relate to HIV pathogenesis is not fully understood. We examined 5185 HIV-1 gp120 nucleotide sequence fragments and clinical data from 154 individuals (152 were infected with HIV-1 Subtype B). Sequences were aligned, translated, manually edited and separated into V1V2, C2, V3, C3, V4, C4 and V5 subregions. V1-V5 and subregion lengths were calculated, and potential N-linked glycosylation sites (PNLGS) counted. Loop lengths and PNLGS were examined as a function of time since infection, CD4 count, viral load, and calendar year in cross-sectional and longitudinal analyses. V1V2 length and PNLGS increased significantly through chronic infection before declining in late-stage infection. In cross-sectional analyses, V1V2 length also increased by calendar year between 1984 and 2004 in subjects with early and mid-stage illness. Our observations suggest that there is little selection for loop length at the time of transmission; following infection, HIV-1 adapts to host immune responses through increased V1V2 length and/or addition of carbohydrate moieties at N-linked glycosylation sites. V1V2 shortening during early and late-stage infection may reflect ineffective host immunity. Transmission from donors with chronic illness may have caused the modest increase in V1V2 length observed during the course of the pandemic.     

Autologous neutralizing humoral immunity and evolution of the viral envelope in the course of subtype B human immunodeficiency virus type 1 infection

Most human immunodeficiency virus type 1 (HIV-1)-infected individuals develop an HIV-specific neutralizing antibody (NAb) response that selects for escape variants of the virus. Here, we studied autologous NAb responses in five typical CCR5-using progressors in relation to viral NAb escape and molecular changes in the viral envelope (Env) in the period from seroconversion until after AIDS diagnosis. In sera from three patients, high-titer neutralizing activity was observed against the earliest autologous virus variants, followed by declining humoral immune responses against subsequent viral escape variants. Autologous neutralizing activity was undetectable in sera from two patients. Patients with high-titer neutralizing activity in serum showed the strongest positive selection pressure on Env early in infection. In the initial phase of infection, gp160 length and the number of potential N-linked glycosylation sites (PNGS) increased in viruses from all patients. Over the course of infection, positive selection pressure declined as the NAb response subsided, coinciding with reversions of changes in gp160 length and the number of PNGS. A number of identical amino acid changes were observed over the course of infection in the viral quasispecies of different patients. Our results indicate that although neutralizing autologous humoral immunity may have a limited effect on the disease course, it is an important selection pressure in virus evolution early in infection, while declining HIV-specific humoral immunity in later stages may coincide with reversion of NAb-driven changes in Env.     

Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility

The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others. Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometry-based strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces cross-clade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env's glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.     

Escape from Autologous Neutralizing Antibodies in Acute/Early Subtype C HIV-1 Infection Requires Multiple Pathways

One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus''s ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization.     

Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies

A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for this region in the elicitation of broadly neutralizing antibody responses against HIV-1.     

A signature in HIV-1 envelope leader peptide associated with transition from acute to chronic infection impacts envelope processing and infectivity

Mucosal transmission of the human immunodeficiency virus (HIV) results in a bottleneck in viral genetic diversity. Gnanakaran and colleagues used a computational strategy to identify signature amino acids at particular positions in Envelope that were associated either with transmitted sequences sampled very early in infection, or sequences sampled during chronic infection. Among the strongest signatures observed was an enrichment for the stable presence of histidine at position 12 at transmission and in early infection, and a recurrent loss of histidine at position 12 in chronic infection. This amino acid lies within the leader peptide of Envelope, a region of the protein that has been shown to influence envelope glycoprotein expression and virion infectivity. We show a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. When expressed in the context of other viral proteins, transmitted envelopes with a basic amino acid position 12 were incorporated at higher density into the virus and exhibited higher infectious titers than did non-signature envelopes. These results support the potential utility of using a computational approach to examine large viral sequence data sets for functional signatures and indicate the importance of Envelope expression levels for efficient HIV transmission.     

Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.     

Selection on the human immunodeficiency virus type 1 proteome following primary infection

Typically during human immunodeficiency virus type 1 (HIV-1) infection, a nearly homogeneous viral population first emerges and then diversifies over time due to selective forces that are poorly understood. To identify these forces, we conducted an intensive longitudinal study of viral genetic changes and T-cell immunity in one subject at < or =17 time points during his first 3 years of infection, and in his infecting partner near the time of transmission. Autologous peptides covering amino acid sites inferred to be under positive selection were powerful for identifying HIV-1-specific cytotoxic-T-lymphocyte (CTL) epitopes. Positive selection and mutations resulting in escape from CTLs occurred across the viral proteome. We detected 25 CTL epitopes, including 14 previously unreported. Seven new epitopes mapped to the viral Env protein, emphasizing Env as a major target of CTLs. One-third of the selected sites were associated with epitopic mutational escapes from CTLs. Most of these resulted from replacement with amino acids found at low database frequency. Another one-third represented acquisition of amino acids found at high database frequency, suggesting potential reversions of CTL epitopic sites recognized by the immune system of the transmitting partner and mutation toward improved viral fitness in the absence of immune targeting within the recipient. A majority of the remaining selected sites occurred in the envelope protein and may have been subjected to humoral immune selection. Hence, a majority of the amino acids undergoing selection in this subject appeared to result from fitness-balanced CTL selection, confirming CTLs as a dominant selective force in HIV-1 infection.     

Detailed analysis of CD4+ Th responses to envelope and Gag proteins of simian immunodeficiency virus reveals an exclusion of broadly reactive Th epitopes from the glycosylated regions of envelope

Ag-specific CD4(+) Th cells play a key role in the development, maturation, and maintenance of pathogen-specific humoral and cellular immune responses. To define the fine specificity of broadly reactive Th responses associated with mature immunity in a lentiviral system, we analyzed peptide-specific Th responses in eight macaques chronically infected with a reference live attenuated SIV at 12-14 mo postinoculation. All macaques had stable immunocompetent Th cells at the time of analysis, and a unique array of Th responses to 20-mer overlapping peptides from envelope (Env) and Gag was identified for each macaque, which were then used to define a set of 31 broadly reactive peptide epitopes. Only 5 of the 31 broadly reactive Th epitope peptides mapped to the surface (SU) domain of Env. Interestingly, these were all confined to two conserved nonglycosylated regions toward the carboxyl terminus of SU, suggesting a structural influence of glycosylation on development of Th responses. Gag and the Env transmembrane proteins contained the majority of broadly reactive peptide epitopes (12 and 14 peptides, respectively), which were uniformly distributed throughout their sequence. This study defines for the first time broadly reactive Th epitope peptides of SIV Env and Gag proteins that are associated with enduring broadly protective vaccine immunity to attenuated SIV, which may be used for the design and evaluation of experimental vaccines. Moreover, the data suggest that extensive glycosylation of SU may provide yet another immune escape mechanism developed by lentiviruses to restrict the breadth of Th repertoire to SU, a major immunologically exposed protein of the virus.     

Characterization of neutralizing antibody responses elicited by clade A envelope immunogens derived from early transmitted viruses

The vast majority of studies with candidate immunogens based on the human immunodeficiency virus envelope (Env) have been conducted with Env proteins derived from clade B viruses isolated during chronic infection. Whether non-clade B Env protein immunogens will elicit antibodies with epitope specificities that are similar to those of antibodies elicited by clade B Envs and whether the antibodies elicited by Envs derived from early transmitted viruses will be similar to those elicited by Envs derived from viruses isolated during chronic infection are currently unknown. Here we performed immunizations with four clade A Envs, cloned directly from the peripheral blood of infected individuals during acute infection, which differed in lengths and extents of glycosylation. The antibody responses elicited by these four Envs were compared to each other and to those elicited by a well-characterized clade B Env immunogen derived from the SF162 virus, which was isolated during chronic infection. Only one clade A Env, the one with the fewer glycosylation sites, elicited homologous neutralizing antibodies (NAbs); these did not target the V1, V2, or V3 regions. In contrast, all four clade A Envs elicited anti-V3 NAbs against "easy-to-neutralize" clade B and clade A isolates, irrespective of the variable region length and extent of glycosylation of the Env used as an immunogen. These anti-V3 NAbs did not access their epitopes on homologous and heterologous clade A, or B, neutralization-resistant viruses. The length and extent of glycosylation of the variable regions on the clade A Env immunogens tested did not affect the breadth of the elicited NAbs. Our data also indicate that the development of cross-reactive NAbs against clade A viruses faces similar hurdles to the development of cross-reactive anti-clade B NAbs.     

Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model

The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (delta5G). In delta5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and delta5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in delta5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in delta5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.     

Hepatitis C virus immune escape via exploitation of a hole in the T cell repertoire

Hepatitis C virus (HCV) infection frequently persists despite eliciting substantial virus-specific immune responses. Thus, HCV infection provides a setting in which to investigate mechanisms of immune escape that allow for viral persistence. Viral amino acid substitutions resulting in decreased MHC binding or impaired Ag processing of T cell epitopes reduce Ag density on the cell surface, permitting evasion of T cell responses in chronic viral infection. Substitutions in viral epitopes that alter TCR contact residues frequently result in escape, but via unclear mechanisms because such substitutions do not reduce surface presentation of peptide-MHC complexes and would be expected to prime T cells with new specificities. We demonstrate that a known in vivo HCV mutation involving a TCR contact residue significantly diminishes T cell recognition and, in contrast to the original sequence, fails to effectively prime naive T cells. This mutant epitope thus escapes de novo immune recognition because there are few highly specific cognate TCR among the primary human T cell repertoire. This example is the first on viral immune escape via exploitation of a "hole" in the T cell repertoire, and may represent an important general mechanism of viral persistence.     

Enhanced Fusion and Virion Incorporation for HIV-1 Subtype C Envelope Glycoproteins with Compact V1/V2 Domains

In infected people, the HIV-1 envelope glycoprotein (Env) constantly evolves to escape the immune response while retaining the essential elements needed to mediate viral entry into target cells. The extensive genetic variation of Env is particularly striking in the V1/V2 hypervariable domains. In this study, we investigated the trade-off, in terms of fusion efficiency, for encoding V1/V2 domains of different lengths. We found that natural variations in V1/V2 length exert a profound impact on HIV-1 entry. Variants encoding compact V1/V2 domains mediated fusion with higher efficiencies than related Envs encoding longer V1/V2 domains. By exchanging the V1/V2 domains between Envs of the same infected person or between two persons linked by a transmission event, we further demonstrated that V1/V2 domains critically influence both Env incorporation into viral particles and fusion to primary CD4 T cells and monocyte-derived dendritic cells. Shortening the V1/V2 domains consistently increased Env incorporation and fusion, whereas lengthening the V1/V2 domains decreased Env incorporation and fusion. Given that in a new host transmitted founder viruses are distinguished by compact Envs with fewer glycosylation sites, our study points to fusion and possibly Env incorporation into virions as limiting steps for transmission of HIV-1 to a new host and suggests that the length and/or the N-glycosylation profile of the V1/V2 domain influences these early steps in the HIV life cycle.     

Failure of innate and adaptive immune responses in controlling hepatitis C virus infection

Effective innate and adaptive immune responses are essential for the control of hepatitis C virus (HCV) infection. Indeed, elimination of HCV during acute infection correlates with an early induction of innate and a delayed induction of adaptive immune responses. However, in the majority of acutely HCV-infected individuals, these responses are insufficient to clear the virus and persistence develops. In recent years, different mechanisms responsible for the failure of innate and adaptive immune responses have been identified. These include the proteolytic cleavage of molecules playing key roles in the induction of the interferon response, manipulation of interferon-induced effector proteins, interference with CD8+ T-cell function or immune escape in T- and B-cell epitopes. In this review, we discuss the possible roles of innate and adaptive immune responses in HCV clearance and the different evasion strategies used by the virus to escape these immune responses.     

Unique mutational patterns in the envelope alpha 2 amphipathic helix and acquisition of length in gp120 hypervariable domains are associated with resistance to autologous neutralization of subtype C human immunodeficiency virus type 1

Autologous neutralizing antibodies (NAb) against human immunodeficiency virus type 1 generate viral escape variants; however, the mechanisms of escape are not clearly defined. In a previous study, we determined the susceptibilities of 48 donor and 25 recipient envelope (Env) glycoproteins from five subtype C heterosexual transmission pairs to NAb in donor plasma by using a virus pseudotyping assay, thereby providing an ideal setting to probe the determinants of susceptibility to neutralization. In the present study, acquisition of length in the Env gp120 hypervariable domains was shown to correlate with resistance to NAb in donor plasma (P = 0.01; Kendall's tau test) but not in heterologous plasma. Sequence divergence in the gp120 V1-to-V4 region also correlated with resistance to donor (P = 0.0002) and heterologous (P = 0.001) NAb. A mutual information analysis suggested possible associations of nine amino acid positions in V1 to V4 with NAb resistance to the donor's antibodies, and five of these were located within an 18-residue amphipathic helix (alpha2) located on the gp120 outer domain. High nonsynonymous-to-synonymous substitution (dN/dS) ratios, indicative of positive selection, were also found at these five positions in subtype C sequences in the database. Nevertheless, exchange of the entire alpha2 helix between resistant donor Envs and sensitive recipient Envs did not alter the NAb phenotype. The combined mutual information and dN/dS analyses suggest that unique mutational patterns in alpha2 and insertions in the V1-to-V4 region are associated with NAb resistance during subtype C infection but that the selected positions within the alpha2 helix must be linked to still other changes in Env to confer antibody escape. These findings suggest that subtype C viruses utilize mutations in the alpha2 helix for efficient viral replication and immune avoidance.     

Serotypic analysis of VP3 and VP7 neutralization escape mutants of rhesus rotavirus.

Neutralization escape mutants of simian rotaviruses (rhesus rotavirus and SA11) were tested in hemagglutination inhibition and neutralization assays against hyperimmune and infection sera to determine if mutation in an immunodominant epitope could enable neutralization escape. An SA11 mutant with a new glycosylation site at amino acid 211 of VP7 was shown to escape neutralization by hyperimmune but not infection sera.     

Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein

A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.     

Altering effects of antigenic variations in HIV-1 on antiviral effectiveness of HIV-specific CTLs

The mutational escape of HIV-1 from established CTL responses is becoming evident. However, it is not yet clear whether antigenic variations of HIV-1 may have an additional effect on the differential antiviral effectiveness of HIV-specific CTLs. Herein, we characterized HIV-specific CTL responses toward Pol, Env, and Nef optimal epitopes presented by HLA-B*35 during a chronic phase of HIV-1 infection. We found CTL escape variants within Pol and Nef epitopes that affected recognition by TCRs, although there was no mutation within the Env epitope. An analysis of peptide-HLA tetrameric complexes revealed that CD8 T cells exclusively specific for the Nef variant were generated following domination by the variant viruses. The variant-specific cells were capable of killing target cells and producing antiviral cytokines but showed impaired Ag-specific proliferation ex vivo, whereas wild-type specific cells had potent activities. Moreover, clonotypic CD8 T cells specific for the Pol variant showed diminished proliferation, whereas Env-specific ones had no functional heterogeneity. Taken together, our data indicate that antigenic variations that abolished TCR recognition not only resulted in escape from established CTL responses but also eventually generated another subset of variant-specific CTLs having decreased antiviral activity, causing an additional negative effect on antiviral immune responses during a chronic HIV infection.     

Interaction of human, rat, and mouse immunoglobulin A (IgA) with Staphylococcal superantigen-like 7 (SSL7) decoy protein and leukocyte IgA receptor

Host survival depends on an effective immune system and pathogen survival on the effectiveness of immune evasion mechanisms. Staphylococcus aureus utilizes a number of molecules to modulate host immunity, including the SSL family of which SSL7 binds IgA and inhibits Fcα receptor I (FcαRI)-mediated function. Other Gram-positive bacterial pathogens produce IgA binding proteins, which, similar to SSL7, also bind the Fc at the CH2/CH3 interface (the junction between constant domains 2 and 3 of the heavy chain). The opposing activities of the host FcαRI-IgA receptor ligand pair and the pathogen decoy proteins select for host and pathogen variants, which exert stronger protection or evasion, respectively. Curiously, mouse but not rat IgA contains a putative N-linked glycosylation site in the center of this host receptor and pathogen-binding site. Here, we demonstrate that this site is glycosylated and that the effect of amino acid changes and glycosylation of the CH2/CH3 interface inhibits interaction with the pathogen IgA binding protein SSL7, while maintaining binding of pIgR, essential to the biosynthesis and transport of SIgA.