Human cytomegalovirus neutralizing antibody-resistant phenotype is associated with reduced expression of glycoprotein H.

We have characterized a neutralizing antibody-resistant mutant human cytomegalovirus (HCMV) obtained from a patient treated with a human monoclonal antiglycoprotein H (gH; unique long region 75) antibody. This virus exhibited resistance to several different neutralizing anti-gH murine monoclonal antibodies (MAbs), as well as to a polyvalent anti-gH serum. The resistant phenotype was unstable and could be maintained only by passage of plaque-purified virus under neutralizing MAb selection. In the absence of a MAb, the resistant phenotype reverted to a neutralizing antibody-sensitive phenotype within one passage. The predicted amino acid sequences of gH from the MAb-resistant and -susceptible parent viruses were identical. Biochemical analysis of the MAb-resistant and -susceptible parent viruses revealed a marked decrease of gH expression in the envelope of the MAb-resistant virus. Furthermore, propagation of the virus in various MAb concentrations resulted in the production of extracellular virions with various levels of resistance to the neutralizing activity of the MAb. These results suggest a mechanism for the generation of neutralizing antibody-resistant viruses which could evade host-derived antiviral antibody responses. In addition, our findings indicate that the stoichiometry of gH in the envelope of infectious HCMV virions is not rigidly fixed and therefore offer a simple explanation for production of phenotypic variants of HCMV through an assembly process in which the content of gH in the envelope of progeny virions varies randomly.     

Blocking antibody access to neutralizing domains on glycoproteins involved in entry as a novel mechanism of immune evasion by herpes simplex virus type 1 glycoproteins C and E

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins.     

Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.

The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.     

The genes encoding the gCIII complex of human cytomegalovirus exist in highly diverse combinations in clinical isolates

The UL74 (glycoprotein O [gO])-UL75 (gH)-UL115 (gL) complex of human cytomegalovirus (CMV), known as the gCIII complex, is likely to play an important role in the life cycle of the virus. The gH and gL proteins have been associated with biological activities, such as the induction of virus-neutralizing antibody, cell-virus fusion, and cell-to-cell spread of the virus. The sequences of the two gH gene variants, readily recognizable by restriction endonuclease polymorphism, are well conserved among clinical isolates, but nothing is known about the sequence variability of the gL and gO genes. Sequencing of the full-length gL and gO genes was performed with 22 to 39 clinical isolates, as well as with laboratory strains AD169, Towne, and Toledo, to determine phylogenetically based variants of the genes. The sequence information provided the basis for identifying gL and gO variants by restriction endonuclease polymorphism. The predicted gL amino acid sequences varied less than 2% among the isolates, but the variability of gO among the isolates approached 45%. The variants of the genes coding for gCIII in laboratory strains Towne, AD169, and Toledo were different from those in most clinical isolates. When clinical isolates from different patient populations with various degrees of symptomatic CMV disease were surveyed, the gO1 variant occurred almost exclusively with the gH1 variant. The gL2 variant occurred with a significantly lower frequency in the gH1 variant group. There were no configurations of the gCIII complex that were specifically associated with symptomatic CMV disease or human immunodeficiency virus serologic status. The potential for the gCIII complex to exist in diverse genetic combinations in clinical isolates points to a new aspect that must be considered in studies of the significance of CMV strain variability.     

Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay.

Two mutations affecting herpes simplex virus type 1 glycoprotein B were mapped by marker rescue using cloned sequences of wild-type herpes simplex virus type 1 strain KOS DNA. One mutant, tsB5, is a temperature-sensitive mutant which does not express mature, functional glycoprotein B at the nonpermissive temperature. The other mutant, marB1.1, expresses an antigenic variant of glycoprotein B and was selected for resistance to neutralization by a monoclonal antibody. The mutation in tsB5 mapped to a 1.2-kilobase segment of the herpes simplex virus type 1 genome between coordinates 0.361 and 0.368, whereas the mutation in marB1.1 mapped to a 1.6-kilobase segment between coordinates 0.350 and 0.361. An in situ enzyme immunoassay was used to detect plaques of recombinant wild-type virus among the progeny of transfections with mutant marB1.1 DNA and wild-type DNA fragments.     

Increased sensitivity to CD4 binding site-directed neutralization following in vitro propagation on primary lymphocytes of a neutralization-resistant human immunodeficiency virus IIIB strain isolated from an accidentally infected laboratory worker

We previously described the adaptation of the neutralization-sensitive human immunodeficiency virus type 1 (HIV-1) strain IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker. During long-term propagation of this resistant isolate, designated FF3346, on primary peripheral blood leukocytes in vitro, an HIV-1 variant appeared that had regained sensitivity to neutralization by soluble CD4 (sCD4) and the broadly neutralizing monoclonal antibody b12. When an early passage of FF3346 was subjected to limiting-dilution culture in peripheral blood mononuclear cells, eight virus variants with various degrees of neutralization resistance were isolated. Two of them, the sCD4 neutralization-resistant variant LW_H8(res) and the sCD4 neutralization-sensitive variant LW_G9(sens), were selected for further study. Interestingly, these two viruses were equally resistant to neutralization by agents that recognize domains other than the CD4 binding site. Site-directed mutagenesis revealed that the increased neutralization sensitivity of variant LW_G9(sens) resulted from only two changes, an Asn-to-Ser substitution at position 164 in the V2 loop and an Ala-to-Glu substitution at position 370 in the C3 domain of gp120. In agreement with this notion, the affinity of b12 for monomeric gp120 containing the N164S and A370E substitutions in the background of the molecular clone LW_H8(res) was higher than its affinity for the parental gp120. Surprisingly, no correlation was observed between CD4 binding affinity for monomeric gp120 and the level of neutralization resistance, suggesting that differences in sCD4 neutralization sensitivity between these viruses are only manifested in the context of the tertiary or quaternary structure of gp120 on the viral surface. The results obtained here indicate that the neutralization-sensitive strain IIIB can become neutralization resistant in vivo under selective pressure by neutralizing antibodies but that this resistance may be easily reversed in the absence of immunological pressure.     

Mutagenic stabilization and/or disruption of a CD4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.     

Structural and Antigenic Analysis of a Truncated Form of the Herpes Simplex Virus Glycoprotein gH-gL Complex

The herpes simplex virus (HSV) gH-gL complex is essential for virus infectivity and is a major antigen for the host immune system. The association of gH with gL is required for correct folding, cell surface trafficking, and membrane presentation of the complex. Previously, a mammalian cell line was constructed which produces a secreted form of gHt-gL complex lacking the transmembrane and cytoplasmic tail regions of gH. gHt-gL retains a conformation similar to that of its full-length counterpart in HSV-infected cells. Here, we examined the structural and antigenic properties of gHt-gL. We first determined its stoichiometry and carbohydrate composition. We found that the complex consists of one molecule each of gH and gL. The N-linked carbohydrate (N-CHO) site on gL and most of the N-CHO sites on gH are utilized, and both proteins also contain O-linked carbohydrate and sialic acid. These results suggest that the complex is processed to the mature form via the Golgi network prior to secretion. To determine the antigenically active sites of gH and gL, we mapped the epitopes of a panel of gH and gL monoclonal antibodies (MAbs), using a series of gH and gL C-terminal truncation variant proteins produced in transiently transfected mammalian cells. Sixteen gH MAbs (including H6 and 37S) reacted with the N-terminal portion of gH between amino acids 19 and 276. One of the gH MAbs, H12, reacted with the middle portion of gH (residues 476 to 678). Nine gL MAbs (including 8H4 and VIII 62) reacted with continuous epitopes within the C-terminal portion of gL, and this region was further mapped within amino acids 168 to 178 with overlapping synthetic peptides. Finally, plasmids expressing the gH and gL truncations were employed in cotransfection assays to define the minimal regions of both gH and gL required for complex formation and secretion. The first 323 amino acids of gH and the first 161 amino acids of gL can form a stable secreted hetero-oligomer with gL and gH792, respectively, while gH323-gL168 is the smallest secreted hetero-oligomer. The first 648 amino acids of gH are required for reactivity with MAbs LP11 and 53S, indicating that a complex of gH648-gL oligomerizes into the correct conformation. The data suggest that both antigenic activity and oligomeric structure require the amino-terminal portions of gH and gL.     

Selection of influenza A virus adsorptive mutants by growth in the presence of a mixture of monoclonal antihemagglutinin antibodies.

The influenza virus hemagglutinin contains four major regions that are recognized by antibodies able to neutralize viral infectivity. To investigate the effect of an antibody response directed against each of these sites on viral evolution, influenza virus A/PR/8/34 (H1N1) was grown in allantois-on-shell cultures in the presence of a mixture of monoclonal antihemagglutinin antibodies. This selection mixture contained antibodies (two or three antibodies per antigenic site) whose concentrations were adjusted to achieve equal neutralization titers against each of the four antigenic sites. By varying the ratio of input virus to selection mixture concentration, we observed that variant viruses emerged under conditions of partial neutralization. Each of the four variants characterized in detail differed from the parental virus in its interaction with cellular receptors and exhibited minimal changes in antigenicity. Thus, these variants were virtually indistinguishable from wild-type viruses, as assessed by the binding of 103 monoclonal antihemagglutinin antibodies in an indirect radioimmunoassay. Despite this, many of the same antibodies demonstrated decreased titers to the variants in hemagglutination inhibition tests. The magnitude of the differences depended on the indicator erythrocytes used (much greater differences were detected with chicken erythrocytes than with human erythrocytes). Hemagglutination mediated by the variants was more resistant to neuraminidase treatment of erythrocytes than hemagglutination mediated by the parental virus. These findings are consistent with the idea that the variants were initially selected by virtue of their increased avidity for host cell receptors. Sequencing of viral RNA revealed that each of the variants differed from the parental virus by a single amino acid alteration in its HA1 subunit. Two of the changes were close to the proposed receptor binding site on hemagglutinin and could directly alter receptor binding, while a third was located near the trimer interface and may have increased receptor binding by altering monomer-monomer interactions.     

Glycoprotein N of Human Cytomegalovirus Protects the Virus from Neutralizing Antibodies

Herpes viruses persist in the infected host and are transmitted between hosts in the presence of a fully functional humoral immune response, suggesting that they can evade neutralization by antiviral antibodies. Human cytomegalovirus (HCMV) encodes a number of polymorphic highly glycosylated virion glycoproteins (g), including the essential envelope glycoprotein, gN. We have tested the hypothesis that glycosylation of gN contributes to resistance of the virus to neutralizing antibodies. Recombinant viruses carrying deletions in serine/threonine rich sequences within the glycosylated surface domain of gN were constructed in the genetic background of HCMV strain AD169. The deletions had no influence on the formation of the gM/gN complex and in vitro replication of the respective viruses compared to the parent virus. The gN-truncated viruses were significantly more susceptible to neutralization by a gN-specific monoclonal antibody and in addition by a number of gB- and gH-specific monoclonal antibodies. Sera from individuals previously infected with HCMV also more efficiently neutralized gN-truncated viruses. Immunization of mice with viruses that expressed the truncated forms of gN resulted in significantly higher serum neutralizing antibody titers against the homologous strain that was accompanied by increased antibody titers against known neutralizing epitopes on gB and gH. Importantly, neutralization activity of sera from animals immunized with gN-truncated virus did not exhibit enhanced neutralizing activity against the parental wild type virus carrying the fully glycosylated wild type gN. Our results indicate that the extensive glycosylation of gN could represent a potentially important mechanism by which HCMV neutralization by a number of different antibody reactivities can be inhibited.     

Selection for Neutralization Resistance of the Simian/Human Immunodeficiency Virus SHIVSF33A Variant In Vivo by Virtue of Sequence Changes in the Extracellular Envelope Glycoprotein That Modify N-Linked Glycosylation

We previously reported on the in vivo adaptation of an infectious molecular simian/human immunodeficiency virus (SHIV) clone, SHIVSF33, into a pathogenic biologic viral variant, designated SHIVSF33A. In the present study, we show that SHIVSF33A is resistant to neutralization by human immunodeficiency virus (HIV) and SHIV antisera. Multiple amino acid substitutions accumulated over time throughout the env gene of SHIVSF33A; some of them coincided with the acquisition of the neutralization resistance of the virus. Of interest are changes that resulted in the removal, repositioning, and addition of potential glycosylation sites within the V1, V2, and V3 regions of envelope gp120. To determine whether potential glycosylation changes within these principal neutralization domains of HIV type 1 formed the basis for the resistance to serum neutralization of SHIVSF33A, mutant viruses were generated on the backbone of parental SHIVSF33 and tested for their neutralization sensitivity. The mutations generated did not alter the in vitro replication kinetics or cytopathicity of the mutant viruses in T-cell lines. However, the removal of a potential glycosylation site in the V1 domain or the creation of such a site in the V3 domain did allow the virus to escape serum neutralization antibodies that recognized parental SHIVSF33. The combination of the V1 and V3 mutations conferred an additive effect on neutralization resistance over that of the single mutations. Taken together, these data suggest that (i) SHIV variants with changes in the Env SU can be selected in vivo primarily by virtue of their ability to escape neutralizing antibody recognition and (ii) carbohydrates play an important role in conferring neutralization escape, possibly by altering the structure of envelope gp120 or by shielding principal neutralization sites.     

Influence of the mutant spectrum in viral evolution: focused selection of antigenic variants in a reconstructed viral quasispecies

RNA viruses replicate as complex mutant distributions termed viral quasispecies. Despite this, studies on virus populations subjected to positive selection have generally been performed and analyzed as if the viral population consisted of a defined genomic nucleotide sequence; such a simplification may not reflect accurately the molecular events underlying the selection process. In the present study, we have reconstructed a foot-and-mouth disease virus quasispecies with multiple, low-frequency, genetically distinguishable mutants that can escape neutralization by a monoclonal antibody. Some of the mutants included an amino acid substitution that affected an integrin recognition motif that overlaps with the antibody-binding site, whereas other mutants included an amino acid substitution that affected antibody binding but not integrin recognition. We have monitored consensus and clonal nucleotide sequences of populations passaged either in the absence or the presence of the neutralizing antibody. In both cases, the populations focused toward a specific mutant that was surrounded by a cloud of mutants with different antigenic and cell recognition specificities. In the absence of antibody selection, an antigenic variant that maintained integrin recognition became dominant, but the mutant cloud included as one of its minority components a variant with altered integrin recognition. Conversely, in the presence of antibody selection, a variant with altered integrin recognition motif became dominant, but it was surrounded by a cloud of antigenic variants that maintained integrin recognition. The results have documented that a mutant spectrum can exert an influence on a viral population subjected to a sustained positive selection pressure and have unveiled a mechanism of antigenic flexibility in viral populations, consisting in the presence in the selected quasispecies of mutants with different antigenic and cell recognition specificities.     

Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies

The ability of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) specific human monoclonal antibodies (MAbs) b12, 2G12, 2F5, and 4E10 to neutralize recently transmitted viruses has not yet been explored in detail. We investigated the neutralization sensitivity of subtype B HIV-1 variants obtained from four primary HIV infection cases and six transmission couples (four homosexual and two parenteral) to these MAbs. Sexually transmitted HIV-1 variants isolated within the first 2 months after seroconversion were generally sensitive to 2F5, moderately resistant to 4E10 and b12, and initially resistant but later more sensitive to 2G12 neutralization. In the four homosexual transmission couples, MAb neutralization sensitivity of HIV in recipients did not correlate with the MAb neutralization sensitivity of HIV from their source partners, whereas the neutralization sensitivity of donor and recipient viruses involved in parenteral transmission was more similar. For a fraction (11%) of the HIV-1 variants analyzed here, neutralization by 2G12 could not be predicted by the presence of N-linked glycosylation sites previously described to be involved in 2G12 binding. Resistance to 2F5 and 4E10 neutralization did also not correlate with mutations in the respective core epitopes. Overall, we observed that the neutralization resistance of recently transmitted subtype B HIV-1 variants was relatively high. Although 8 of 10 patients had viruses that were sensitive to neutralization by at least one of the four broadly neutralizing antibodies studied, 4 of 10 patients harbored at least one virus variant that seemed resistant to all four antibodies. Our results suggest that vaccine antigens that only elicit antibodies equivalent to b12, 2G12, 2F5, and 4E10 may not be sufficient to protect against all contemporary HIV-1 variants and that additional cross-neutralizing specificities need to be sought.     

Glycosylation of gp41 of simian immunodeficiency virus shields epitopes that can be targets for neutralizing antibodies

Human immunodeficiency virus type 1 and simian immunodeficiency virus possess three closely spaced, highly conserved sites for N-linked carbohydrate attachment in the extracellular domain of the transmembrane protein gp41. We infected rhesus monkeys with a variant of cloned SIVmac239 lacking the second and third sites or with a variant strain lacking all three of SIVmac239's glycosylation sites in gp41. For each mutation, asparagine (N) in the canonical N-X-S/T recognition sequence for carbohydrate attachment was changed to the structurally similar glutamine such that two nucleotide changes would be required for a reversion of the mutated codon. By 16 weeks, experimentally infected monkeys made antibodies that neutralized the mutant viruses to high titers. Such antibodies were not observed in monkeys infected with the parental virus. Thus, new specificities were revealed as a result of the carbohydrate attachment mutations, and antibodies of these specificities had neutralizing activity. Unlike monkeys infected with the parental virus, monkeys infected with the mutant viruses made antibodies that reacted with peptides corresponding to the sequences in this region. Furthermore, there was strong selective pressure for the emergence of variant sequences in this region during the course of infection. By analyzing the neutralization profiles of sequence variants, we were able to define three mutations (Q625R, K631N, and Q634H) in the region of the glycosylation site mutations that conferred resistance to neutralization by plasma from the monkeys infected with mutant virus. Based on the reactivity of antibodies to peptides in this region and the colocalization of neutralization escape mutations, we conclude that N-linked carbohydrates in the ectodomain of the transmembrane protein shield underlying epitopes that would otherwise be the direct targets of neutralizing antibodies.     

A panel of IgG1 b12 variants with selectively diminished or enhanced affinity for Fcγ receptors to define the role of effector functions in protection against HIV

Passive transfer of neutralizing antibodies is effective in protecting rhesus macaques against simian/human immunodeficiency virus (SHIV) challenge. In addition to neutralization, effector functions of the crystallizable fragment (Fc) of antibodies are involved in antibody-mediated protection against a number of viruses. We recently showed that interaction between the Fc fragment of the broadly neutralizing antibody IgG1 b12 and cellular Fcγ receptors (FcγRs) plays an important role in protection against SHIV infection in rhesus macaques. The specific nature of this Fc-dependent protection is largely unknown. To investigate, we generated a panel of 11 IgG1 b12 antibody variants with selectively diminished or enhanced affinity for the two main activating FcγRs, FcγRIIa and FcγRIIIa. All 11 antibody variants bind gp120 and neutralize virus as effectively as does wild-type b12. Binding studies using monomeric (enzyme-linked immunosorbent assay [ELISA] and surface plasmon resonance [SPR]) and cellularly expressed Fcγ receptors show decreased (up to 5-fold) and increased (up to 90-fold) binding to FcγRIIa and FcγRIIIa with this newly generated panel of antibodies. In addition, there was generally a good correlation between b12 variant affinity for Fcγ receptor and variant function in antibody-dependent cell-mediated virus inhibition (ADCVI), phagocytosis, NK cell activation assays, and antibody-dependent cellular cytotoxicity (ADCC) assays. In future studies, these b12 variants will enable the investigation of the protective role of individual FcγRs in HIV infection.     

Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees.

Emergence in two chimpanzees of human immunodeficiency virus type 1 (HIV-1) IIIB variants resistant to neutralization by the preexisting antibody is described. Viruses isolated from the HIV-1 IIIB gp120-vaccinated and -challenged animal were more resistant to neutralization by the chimpanzee's own serum than viruses isolated from the naive infected animal, indicating immune pressure as the selective mechanism. However, all reisolated viruses were 16- to 256-fold more neutralization resistant than the inoculum virus to antibodies binding to the third variable domain (V3) of the HIV-1 external envelope. Early chimpanzee serum samples that neutralized the inoculum strain but not the reisolated viruses were found to bind an HIV-1 IIIB common nonapeptide (IQRGPGRAF) derived from the gp120 isolate-specific V3 domain shown to induce isolate-specific neutralization in other animals. Amplification of the V3 coding sequence by polymerase chain reaction and subsequent sequence analysis of the neutralization-resistant variants obtained from in vivo-infected animals indicated that early resistance to neutralization by an HIV-1 IIIB monoclonal antibody (0.5 beta) was conferred by changes outside the direct binding site for the selective neutralizing antibody. The reisolated neutralization-resistant isolates consisted of the lower-replication-competent virus subpopulations of the HIV-1 IIIB stock, as confirmed by biological and sequence analyses. In vitro passage of the HIV-1 IIIB stock through chimpanzee and human peripheral blood mononuclear cell cultures void of HIV-specific antibody resulted in homogenic amplification of the more-replication-competent subpopulation preexisting in the original viral stock, suggesting a role for the immune system in suppressing the more-replication-competent viruses.     

Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. off.

IgG1b12, a human monoclonal antibody (MAb) to an epitope overlapping the CD4-binding site on gp120, has broad and potent neutralizing activity against most primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess whether and how escape mutants resistant to IgG1b12 can be generated, we cultured primary HIV-1 strain JRCSF in its presence. An escape mutant emerged which was approximately 100-fold more resistant to neutralization by IgG1b12. Both virion-associated and solubilized gp120 from this variant had a reduced affinity for IgG1b12, and sequencing of its env gene showed that amino acid substitutions had occurred at three positions within gp120. Two (D164N and D182N) were located in V2, and one (P365L) was in C3. By site-directed mutagenesis, we demonstrated that the D182N and P365L mutations, but not D164N, contribute to the IgG1b12-resistant phenotype. However, the former two substitutions, individually or in combination, hinder the replication of the neutralization-resistant virus. Introduction of the D164N substitution into the P365L variant results in a nonviable virus (D164N/P365L). In contrast, addition of D164N to the D182N or D182N/P365L mutant partially restored replicative function to near wild-type levels. Furthermore, we found that all of the IgG1b12-resistant mutant viruses remained sensitive to other human MAbs, such as 2G12 and 2F5, and to the CD4-IgG molecule, except that the P365L-containing mutant was slightly resistant to CD4-IgG. These results suggest that escape from IgG1b12 neutralization is due to a local rather than a global modification of the gp120 structure. Our findings have implications for the therapeutic and prophylactic applications of antibodies for HIV-1 infection.     

Vaccination with SARS-CoV-2 variants of concern protects mice from challenge with wild-type virus

Vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against Coronavirus Disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild-type SARS-CoV-2 and from variants B.1.1.7, B.1.351, and P.1 for their immunogenicity and protective effect in vivo against challenge with wild-type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7-vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild-type SARS-CoV-2 in a mouse model.

This study explores the immune response induced by wild type and variant SARS-CoV-2 spike proteins, and the protection that these immune responses provide against challenge with wild type virus in the mouse model.     

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

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.     

Antibody Escape Kinetics of Equine Infectious Anemia Virus Infection of Horses

Lentivirus escape from neutralizing antibodies (NAbs) is not well understood. In this work, we quantified antibody escape of a lentivirus, using antibody escape data from horses infected with equine infectious anemia virus. We calculated antibody blocking rates of wild-type virus, fitness costs of mutant virus, and growth rates of both viruses. These quantitative kinetic estimates of antibody escape are important for understanding lentiviral control by antibody neutralization and in developing NAb-eliciting vaccine strategies.