Studies on the neutralization of herpes simplex virus. XI. Differences between slow-reacting complement-requiring neutralizing (s-CRN) antibodies in IgG and IgM

Late IgG and IgM from a rabbit immunized with herpes virus were tested for ordinary neutralizing (N) antibody, complement-requiring neutralizing (CRN) antibody and in addition CRN antibody detectable by overnight sensitization at 0 C (s-CRN antibody). Heat stability tests showed that IgG s-CRN antibody was slightly less resistant to heating at 70 C than were N and CRN antibodies, whereas all three activities of IgM were quickly degraded at this temperature. Dose-response curves with varying amounts of complement (C) or anti-antibody revealed a marked difference between IgG s-CRN and IgM s-CRN antibodies. While 1-hr sensitization at 37 C was insufficient to detect IgG s-CRN antibody, it had the same effect as overnight sensitization at 0 C for IgM s-CRN antibody. When sensitization at 0 C was prolonged to 3 days, unexpectedly high endpoints exceeding 1:10,000 were obtained even with IgM. consequently, enhancement by C was several hundred-fold with IgM in contrast to 5- to 10-fold enhancement of IgG s-CRN antibody, which was similar to that after overnight sensitization. Also IgM obviously required more C than did IgG. These results suggest that IgM of late serum is slower reacting and more C-dependent than IgG s-CRN antibody. Tests with early rabbit sera indicated that the s-CRN antibody detectable by 3-day sensitization reaches a high level before the appearance of N antibody.     

Studies on the neutralization of herpes simplex virus. IX. Variance in complement requirement among IgG and IgM from early and late sera under different sensitization conditions.

Early and late sera of rabbits immunized with herpes simplex virus were fractionated into IgG and IgM, and the minimal concentration of complement (C) required for full enhancement of neutralizing activity was determined for each by the plaque reduction method. In tests employing simultaneous mixing of virus, antibody and C, C-requiring neutralizing (CRN) antibody in IgM required 2–8 times more C than that in IgG. When virus-antibody mixtures were incubated at 0 C overnight before addition of C, a marked enhancement of CRN endopoint especially of late IgG and IgM was exhibited, in contrast to materially unchanged titers of the ordinary neutralizing antibody. This result suggested an abundance of slow-reacting CRN-virus complexes. The CRN antibody so detected required about 4 times more C than that detectable by the usual test in the case of late IgG and IgM. When virus sensitized with late IgG at 0 C overnight was further incubated at 37 C for 1 hr, the C requirement changed but slightly without showing any more increase of the endpoint, whereas sensitization at 0 C for 2 to 3 days further increased the CRN antibody endpoint but the C requirement was equal to that after 1 day's sensitization at 0 C. Based on these and earlier findings, a hypothesis is proposed that binding of a single antibody molecule with virus may cause a series of changes of the virus particle or part of those changes depending on the nature of antibody and on the sensitization condition, and C added to such complexes at an appropriate stage of the changes can accelerate the procession of the changes leading eventually to inactivation.     

Studies on the neutralization of herpes simplex virus. X. Demonstration of complement-requiring neutralizing (CRN) and slow-reacting CRN (s-CRN) antibodies in late IgG.

A sample of late IgG from a rabbit hyperimmunized with herpes simplex virus was analyzed for neutralizing (N) and complement-requiring neutralizing (CRN) antibodies. In a usual endpoint test, N and CRN titers were 1: 40 and 1: 160, respectively, but when virus-IgG mixtures were incubated at 0 C overnight before addition of complement (C), an endpoint of 1:1280 was obtained. Virus sensitized at 0 C overnight required more C for inactivation than did sensitized virus formed earlier. Sensitization kinetic curve experiments employing a proper initial virus concentration, which permitted differentiation of sensitized viruses requiring different amounts of C, indicated that formation of sensitized virus detectable only with a relatively large amount of C proceeded slowly at IgG dilutions where the ordinary CRN antibody requiring a smaller amount of C was negligible. The results strongly suggested that the IgG sample contained slow-reacting CRN (s-CRN) antibody in excess of the hitherto known CRN antibody. As to the mechanism of formation of s-CRN complexes, experiments failed to prove the occurrence of complexes initially insensitive to C, and it appears more likely that s-CRN antibody has a comparatively low avidity for virus.     

Complement requirement of neutralizing antibodies in different classes of immunoglobulin appearing in rabbits and guinea pigs after primary and booster immunizations with herpes simplex virus.

Rabbits and guinea pigs were immunized with herpes simplex virus and bled periodically. The sera were fractionated into slow IgG, fast IgG and IgM by DEAE-cellulose column chromatography, and complement-requiring (CRN) and nonrequiring neutralizing (N) antibody activities were estimated. In early sera of rabbits, the two IgG and IgM fractions possessed about equal CRN activities, although some animals showed a slightly lower activity in fast IgG. In guinea pigs, the early CRN activity resided mainly in slow IgS (7 S gamma2). The early IgG antibody of guinea pigs differed from that of rabbits in that it resembled IgM in resistances to heating at 70 C and to 2-merceptoethanol. The level of CRN IgM antibody in rabbits declined following a peak reached in 2 to 3 weeks, whereas such a decline was never observed in guinea pigs. N IgG antibody was developed a few weeks after the first immunization in rabbits and much retarded in guinea pigs. In both species, booster immunization quickly evoked N antibody in the two IgG fractions and also CRN IgM antibody, but in the case of rabbits the IgM antibody disappeared soon. It is concluded that IgG plays an important role in humoral immunity from the initial stage of the immunization course.     

Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus.

Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Fourteen clonally isolated mutants demonstrated substantial resistance to multiple monoclonal antibodies, including K3-4C8-K3-2F2 and B5-B3. In addition, 13 mutants demonstrated a 10-fold or greater reduction in neutraliztion mediated by polyclonal human antibody. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development.     

Complement Requirement of Neutralizing Antibodies in Different Classes of Immunoglobulin Appearing in Rabbits and Guinea Pigs after Primary and Booster Immunizations with Herpes Simplex Virus

Rabbits and guinea pigs were immunized with herpes simplex virus and bled periodically. The sera were fractionated into slow IgG, fast IgG and IgM by DEAE-cellulose column chromatography, and complement-requiring (CRN) and nonrequiring neutralizing (N) antibody activities were estimated. In early sera of rabbits, the two IgG and IgM fractions possessed about equal CRN activities, although some animals showed a slightly lower activity in fast IgG. In guinea pigs, the early CRN activity resided mainly in slow IgG (7 S γ₂). The early IgG antibody of guinea pigs differed from that of rabbits in that it resembled IgM in resistances to heating at 70 C and to 2-mercaptoethanol. The level of CRN IgM antibody in rabbits declined following a peak reached in 2 to 3 weeks, whereas such a decline was never observed in guinea pigs. N IgG antibody was developed a few weeks after the first immunization in rabbits and much retarded in guinea pigs. In both species, booster immunization quickly evoked N antibody in the two IgG fractions and also CRN IgM antibody, but in the case of rabbits the IgM antibody disappeared soon. It is concluded that IgG plays an important role in humoral immunity from the initial stage of the immunization course.     

Infection of a macrophage-like cell line, P388D1 with reovirus; effects of immune ascitic fluids and monoclonal antibodies on neutralization and on enhancement of viral growth

We studied the effect of antibody on the growth of reovirus, serotypes 1 and 3, in P388D1, a continuous mouse macrophage-like cell line. Enhanced growth of virus was observed when cells were infected in the presence of nonneutralizing monoclonal antibodies or subneutralizing concentrations of either immune ascitic fluids or neutralizing monoclonal antibodies. Both enhancement of viral growth and neutralization were accompanied by an antibody-mediated increase in binding of radiolabeled virus to P388D1 cells. Although neutralization was seen only with monoclonal antibodies directed toward the sigma-1 surface protein of the virus, enhancement was observed with two monoclonal antibodies directed toward other surface proteins. Trypsin treatment of P388D1 cells abrogated enhanced growth of virus mediated by a mouse IgG2a antibody; preincubation with P388D1 with human IgG1 but not IgG2 myeloma proteins also abrogated enhancement by immune ascitic fluid or monoclonal antibody. These observations are compatible with known properties of P388D1 Fc receptors and support the role of the Fc receptor in antibody-mediated infection.     

Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization

We describe here the selection of ultra-potent anti-respiratory syncytial virus (RSV) antibodies for preventing RSV infection. A large number of antibody variants derived from Synagis (palivizumab), an anti-RSV monoclonal antibody that targets RSV F protein, were generated by a directed evolution approach that allowed convenient manipulation of the binding kinetics. Palivizumab variants with about 100-fold slower dissociation rates or with fivefold faster association rates were identified and tested for their ability to neutralize virus in a microneutralization assay. Our data reveal a major differential effect of the association and dissociation rates on the RSV neutralization, particularly for intact antibodies wherein the association rate plays the predominant role. Furthermore, we found that antibody binding valence also plays a critical role in mediating the viral neutralization through a mechanism that is likely unrelated to antibody size or binding avidity. We applied an iterative mutagenesis approach, and thereafter were able to identify palivizumab Fab variants with up to 1500-fold improvement and palivizumab IgG variants with up to 44-fold improvement in the ability to neutralize RSV. These anti-RSV antibodies likely will offer great clinical potential for RSV immunoprophylaxis. In addition, our findings provide insights into engineering potent antibody therapeutics for other disease targets.     

Affinity maturation to improve human monoclonal antibody neutralization potency and breadth against hepatitis C virus

A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development.     

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.     

Monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages.

Antibody-dependent enhancement of virus infection is a process whereby virus-antibody complexes initiate infection of cells via Fc receptor-mediated endocytosis. We sought to investigate antibody-dependent enhancement of feline infectious peritonitis virus infection of primary feline peritoneal macrophages in vitro. Enhancement of infection was assessed, after indirect immunofluorescent-antibody labelling of infected cells, by determining the ratio between the number of cells infected in the presence and absence of virus-specific antibody. Infection enhancement was initially demonstrated by using heat-inactivated, virus-specific feline antiserum. Functional compatibility between murine immunoglobulin molecules and feline Fc receptors was demonstrated by using murine anti-sheep erythrocyte serum and an antibody-coated sheep erythrocyte phagocytosis assay. Thirty-seven murine monoclonal antibodies specific for the nucleocapsid, membrane, or spike proteins of feline infectious peritonitis virus or transmissible gastroenteritis virus were assayed for their ability to enhance the infectivity of feline infectious peritonitis virus. Infection enhancement was mediated by a subset of spike protein-specific monoclonal antibodies. A distinct correlation was seen between the ability of a monoclonal antibody to cause virus neutralization in a routine cell culture neutralization assay and its ability to mediate infection enhancement of macrophages. Infection enhancement was shown to be Fc receptor mediated by blockade of antibody-Fc receptor interaction using staphylococcal protein A. Our results are consistent with the hypothesis that antibody-dependent enhancement of feline infectious peritonitis virus infectivity is mediated by antibody directed against specific sites on the spike protein.     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

Neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement

All normal human sera examined neutralized WS/33 H1N1 influenza virus efficiently by one of two antibody-dependent mechanisms. A minority of the sera contained moderate levels of IgG antibody directed against the viral hemagglutinin that had the ability to directly neutralize the virus. The majority of sera tested contained very low levels of IgG anti-hemagglutinin antibody, which was detectable with a specific ELISA but not by conventional HAI assays. Such IgG antibody was unable to directly neutralize the virus. Studies with agammaglobulinemic serum and with sera depleted of and reconstituted with complement components established essential roles for IgG and the components of the classical complement pathway through C3 for neutralization. The components of the alternative and membrane attack pathways were not needed for neutralization. As anticipated from the requirement for IgG and exclusive mediation of neutralization by the classical pathway, the virus-IgG immune complex activated purified C1. Binding of C3 and C4 to the virus was demonstrated, as was classical pathway-mediated triggering of the alternative pathway, with recruitment of properdin. In addition, the H1N1 influenza virus also directly activated the alternative complement pathway in human serum, leading to C3 and properdin deposition on the viral envelope. Such direct alternative pathway activation also required immunoglobulin. However, the alternative pathway alone was unable to neutralize the virus. Thus, most normal sera examined contain low levels of IgG anti-hemagglutinin antibody, which activate the classical pathway of the complement system and neutralize WS/33 influenza virus by deposition of C3 and C4 on the viral envelope.     

A new microplate neutralization test for typing of herpes simplex virus.

A microplate serum neutralization test for estimation of complement-requiring neutralizing (CRN) antibody was established as the first step for simplification of typing of herpes simplex virus (HSV). When guinea pigs were immunized with type 2 HSV, the late sera could mostly differentiate the types of HSV better than hyperimmune rabbit sera, the CRN titer against the heterologous type 1 HSV being much lower than the homologous titer. Sera of guinea pigs immunized with type 1 HSV showed about the same level of cross reaction against type 2 HSV as did rabbit antisera. Guinea pig sera having minimal levels of cross reaction were selected, and their high dilution (1:160) and complement were added to serial 10-fold dilutions of virus in the microplate titration of virus infectivity. Selective reduction of virus titer by either antiserum could determine the type of HSV. No equivocal intermediate case was found among a number of stock strains including many fresh isolates. The typing result coincided with that determined by a modification of Yang et al's method based on virus titers obtained with Vero and primary chick embryo cells. The typing based on plaquing in chick embryo cells sometimes failed to identify type 1 HSV.     

Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.     

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.     

Hemagglutinin 1-specific immunoglobulin G and Fab molecules mediate postattachment neutralization of influenza A virus by inhibition of an early fusion event

In standard neutralization (STAN), virus and antibody are reacted together before inoculation of target cells, and inhibition of almost any of the processes concerned in the early interaction of virus and cell, including inhibition of virus attachment to cell receptors, can be the cause of neutralization by a particular monoclonal antibody (MAb). To simplify the interpretation of antibody action, we carried out a study of postattachment neutralization (PAN), where virus is allowed to attach to target cells before neutralizing antibody is introduced. We used influenza virus A/PR/8/34 (H1N1) and monoclonal immunoglobulin G (IgG) molecules and their Fabs specific to antigenic sites Sb (tip), Ca2 (loop), and Cb (hinge) of the hemagglutinin 1 (HA1) protein. All IgGs and Fabs gave PAN, although with reduced efficiency compared with STAN. Thus, bivalent binding of antibody was not essential for PAN. By definition, none of these MAbs gave PAN by inhibiting virus attachment, and they did not elute attached virus from the target cell or inhibit endocytosis of virus. However, virus-cell fusion, as demonstrated by R18 fluorescence dequenching or hemolysis of red blood cells, was inhibited in direct proportion to neutralization and in a dose-dependent manner and was thus likely to be responsible for the observed neutralization. However, to get PAN, it was necessary to inhibit the activation of the prefusion intermediate, the earliest known form on the fusion pathway that is created when virus is incubated at pH 5 and 4 degrees C. PAN antibodies may act by binding HA trimers in contact with the cell and/or trimers in the immediate vicinity of the virus-cell contact point and so inhibit the recruitment of additional receptor-HA complexes.     

Interactions of human complement with virus particles containing the Nipah virus glycoproteins

Complement is an innate immune response system that most animal viruses encounter during natural infections. We have tested the role of human complement in the neutralization of virus particles harboring the Nipah virus (NiV) glycoproteins. A luciferase-expressing vesicular stomatitis virus (VSV) pseudotype that contained the NiV fusion (F) and attachment (G) glycoproteins (NiVpp) showed dose- and time-dependent activation of human complement through the alternative pathway. In contrast to our findings with other paramyxoviruses, normal human serum (NHS) alone did not neutralize NiVpp infectivity in vitro, and electron microscopy demonstrated no significant deposition of complement component C3 on particles. This lack of NiVpp neutralization by NHS was not due to a global inhibition of complement pathways, since complement was found to significantly enhance neutralization by antibodies specific for the NiV F and G glycoproteins. Complement components C4 and C1q were necessary but not sufficient by themselves for the enhancement of antibody neutralization. Human complement also enhanced NiVpp neutralization by a soluble version of the NiV receptor EphrinB2, and this depended on components in the classical pathway. The ability of complement to enhance neutralization fell into one of two profiles: (i) anti-F monoclonal antibodies showed enhancement only at high and not low antibody concentrations, and (ii) anti-G monoclonal antibodies and EphrinB2 showed enhancement at both high and very low levels of antibody (e.g., 3.1 ng) or EphrinB2 (e.g., 2.5 ng). Together, these data establish the importance of human complement in the neutralization of particles containing the NiV glycoproteins and will help guide the design of more effective therapeutics that harness the potency of complement pathways.     

Recombinant CD4-selected human immunodeficiency virus type 1 variants with reduced gp120 affinity for CD4 and increased cell fusion capacity.

Variants of molecularly cloned human immunodeficiency virus type 1 (HIV-1) were analyzed following selection for the ability to replicate after exposure to soluble, recombinant CD4 protein (rCD4). Two variants, 4/1 and 16/2, show 8-fold and 16-fold reduced sensitivity to rCD4 neutralization yet remain as sensitive as the parental wild-type (wt) virus to neutralization by rCD4-immunoglobulin G (IgG) chimeric molecules and to inhibition of cellular infection by anti-CD4 antibody. The 4/1 variant is more cytopathic, with faster cell fusion and replication kinetics than the wt virus. The gp120s derived from the 4/1 and 16/2 variants have 3-fold and 30-fold reduced binding affinities to rCD4, respectively. The 4/1 variant exhibits diminished shedding of virion gp120 induced by rCD4. The binding of and neutralization by V3 loop antibodies and other anti-gp120 antibodies is reduced for 4/1 but not for 16/2. Sequence analysis revealed a codon change at amino acid residue 435 in the C4 region of the gp120 of 16/2. This accounts for its rCD4 insensitivity, since the insertion of this mutation in the wt gp120 yields the same phenotype. The 4/1 variant has a codon change in the V3 region of gp120 (amino acid 311), which accounts for its reduced sensitivity to some neutralizing antibodies but not to rCD4. The ready selection of rCD4-resistant variants has obvious relevance for rCD4-based therapeutic stratagems.     

Immunodominance and functional activities of antibody responses to inactivated West Nile virus and recombinant subunit vaccines in mice

Factors controlling the dominance of antibody responses to specific sites in viruses and/or protein antigens are ill defined but can be of great importance for the induction of potent immune responses to vaccines. West Nile virus and other related important human-pathogenic flaviviruses display the major target of neutralizing antibodies, the E protein, in an icosahedral shell at the virion surface. Potent neutralizing antibodies were shown to react with the upper surface of domain III (DIII) of this protein. Using the West Nile virus system, we conducted a study on the immunodominance and functional quality of E-specific antibody responses after immunization of mice with soluble protein E (sE) and isolated DIII in comparison to those after immunization with inactivated whole virions. With both virion and sE, the neutralizing response was dominated by DIII-specific antibodies, but the functionality of these antibodies was almost four times higher after virion immunization. Antibodies induced by the isolated DIII had an at least 15-fold lower specific neutralizing activity than those induced by the virion, and only 50% of these antibodies were able to bind to virus particles. Our results suggest that immunization with the tightly packed E in virions focuses the DIII antibody response to the externally exposed sites of this domain which are the primary targets for virus neutralization, different from sE and isolated DIII, which also display protein surfaces that are cryptic in the virion. Despite its low potency for priming, DIII was an excellent boosting antigen, suggesting novel vaccination strategies that strengthen and focus the antibody response to critical neutralizing sites in DIII.