Alla Kachko

Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity

Using human immune globulins made from antihepatitis C virus (HCV)-positive plasma, we recently identified two antibody epitopes in the E2 protein at residues 412–426 (epitope I) and 434–446 (epitope II). Whereas epitope I is highly conserved among genotypes, epitope II varies. We discovered that epitope I was implicated in HCV neutralization whereas the binding of non-neutralizing antibody to epitope II disrupted virus neutralization mediated by antibody binding at epitope I. These findings suggested that, if this interfering mechanism operates in vivo during HCV infection, a neutralizing antibody against epitope I can be restrained by an interfering antibody, which may account for the persistence of HCV even in the presence of an abundance of neutralizing antibodies. We tested this hypothesis by affinity depletion and peptide-blocking of epitope-II-specific antibodies in plasma of a chronically HCV-infected patient and recombinant E1E2 vaccinated chimpanzees. We demonstrate that, by removing the restraints imposed by the interfering antibodies to epitope-II, neutralizing activity can be revealed in plasma that previously failed to neutralize viral stock in cell culture. Further, cross-genotype neutralization could be generated from monospecific plasma. Our studies contribute to understanding the mechanisms of antibody-mediated neutralization and interference and provide a practical approach to the development of more potent and broadly reactive hepatitis C immune globulins.

New neutralizing antibody epitopes in hepatitis C virus envelope glycoproteins are revealed by dissecting peptide recognition profiles.

One of the greatest challenges to HCV vaccine development is the induction of effective immune responses using recombinant proteins or vectors. In order to better understand which vaccine-induced antibodies contribute to neutralization of HCV the quality of polyclonal anti-E1E2 antibody responses in immunized mice and chimpanzees was assessed at the level of epitope recognition using peptide scanning and neutralization of chimeric 1a/2a, 1b/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype 1a (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc. By functionally dissecting the polyclonal immune responses we identified three new regions important for neutralization within E1 (aa264-318) and E2 (aa448-483 and aa496-515) of the HCV glycoproteins, the third of which (aa496-515) is highly conserved (85-95%) amongst genotypes. Antibodies to aa496-515 were isolated by affinity binding and elution from the serum of a vaccinated chimpanzee and found to specifically neutralize chimeric 1a/2a, 1b/2a and 2a HCVcc. IC50 titres (IgG ng/mL) for the aa496-515 eluate were calculated as 142.1, 239.37 and 487.62 against 1a/2a, 1b/2a and 2a HCVcc, respectively. Further analysis demonstrated that although antibody to this new, conserved neutralization epitope is efficiently induced with recombinant proteins in mice and chimpanzees; it is poorly induced during natural infection in patients and chimpanzees (7 out of 68 samples positive) suggesting the epitope is poorly presented to the immune system in the context of the viral particle. These findings have important implications for the development of HCV vaccines and strategies designed to protect against heterologous viruses. The data also suggest that recombinant or synthetic antigens may be more efficient at inducing neutralizing antibodies to certain epitopes and that screening virally infected patients may not be the best approach for finding new cross-reactive epitopes.

T-cell vaccines that elicit effective immune responses against HCV in chimpanzees may create greater immune pressure for viral mutation

A prime/boost vaccine strategy that transfects antigen-presenting cells using ligand-modified immunoliposomes to efficiently deliver plasmid DNA, followed by boosting with non-replicating recombinant adenovirus was used in chimpanzees to generate HCV-specific memory T-cells. Three chimpanzees (two vaccines, one control) were immunized with immunoliposomes complexed with DNA expressing NS3-NS5B or complexed with empty vector. Animals were boosted with adenovirus expressing NS3-NS5B, or non-recombinant adenovirus (control). Using liposome delivery we were able to obtain specific HCV responses following DNA priming in the chimpanzees. This data and mouse immunization studies confirm this as a more efficient delivery system than direct intramuscular inoculations with naked DNA. Subsequent to the adenovirus boost significant increases in peripheral HCV-specific T-cell responses and intrahepatic IFN-gamma and CD3varepsilon mRNA were also observed in the two vaccinated animals. Following challenge (100 CID(50)) both vaccinated animals showed immediate and significant control of viral replication (peak titers 3.7x10(4) and 9x10(3)IU/mL at weeks 1 and 2), which coincided with increases in HCV-specific T-cell responses. Viral kinetics in the control animal were comparable to historical controls with exponential increases in titer during the first several weeks. One vaccinated animal developed a low-level persistent infection (2x10(3)IU/mL) which correlated with a decrease in HCV-specific T-cell responses. Circulating virus isolated from both vaccinated animals showed approximately 2-fold greater nonsynonymous mutation rates compared to controls and the nonsynonymous/synonymous mutation rate ratio was indicative of positive selection. These data suggest that although T-cell vaccines can induce immune responses capable of controlling HCV, they also induce high levels of immune pressure for the potential selection of escape mutants.

Amino acid residue-specific neutralization and nonneutralization of hepatitis C virus by monoclonal antibodies to the E2 protein.

ABSTRACT Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W437, L438, L441, and F442, within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.

Genetic factors of Ebola virus virulence in guinea pigs

Zaire ebolavirus (ZEBOV) causes severe hemorrhagic fever in primates, whereas in guinea pigs it induces a nonlethal infection with a mild fever and subsequent recovery. We performed 7 selective passages in guinea pigs resulted in obtaining of guinea pig-adapted strain (GPA-P7) strain. By the 7th passage, the infection with EBOV induced a lethal disease in animals accompanied by the characteristic hematological changes: leukocytosis (primarily due to neutrophilia) as well as pronounced deficiencies in platelets, lymphocytes, monocytes and significant decrease of blood neutrophils phagocytic capacity. Increasing of virulence correlated with appearance of several nucleotide substitutions: in the genes NP, A2166G (N566S), VP24, U10784C (L147P), G10557A (M71I), G10805U (R154L), and L, G12286A (V236I). It has been theoretically calculated that the mutations associated with an increase in EBOV virulence can confer characteristic secondary structure on the proteins NP (C-terminal region) and full-sized VP24.

Inhibition of Hepatitis C Virus by the Cyanobacterial Protein Microcystis viridis Lectin: Mechanistic Differences between the High-Mannose Specific Lectins MVL, CV-N, and GNA

Plant or microbial lectins are known to exhibit potent antiviral activities against viruses with glycosylated surface proteins, yet the mechanism(s) by which these carbohydrate-binding proteins exert their antiviral activities is not fully understood. Hepatitis C virus (HCV) is known to possess glycosylated envelope proteins (gpE1E2) and to be potently inhibited by lectins. Here, we tested in detail the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture HCV, as well as their binding properties toward viral particles, target cells, and recombinant HCV glycoproteins. Using infectivity assays, CV-N, MVL, and GNA inhibited HCV with IC50 values of 0.6 nM, 30.4 nM, and 11.1 nM, respectively. Biolayer interferometry analysis demonstrated a higher affinity of GNA to immobilized recombinant HCV glycoproteins compared to CV-N and MVL. Complementary studies, including fluorescence-activated cell sorting (FACS) analysis, confocal microscopy, and pre- and post-virus binding assays, showed a complex mechanism of inhibition for CV-N and MVL that includes both viral and cell association, while GNA functions by binding directly to the viral particle. Combinations of GNA with CV-N or MVL in HCV infection studies revealed synergistic inhibitory effects, which can be explained by different glycan recognition profiles of the mainly high-mannoside specific lectins, and supports the hypothesis that these lectins inhibit through different and complex modes of action. Our findings provide important insights into the mechanisms by which lectins inhibit HCV infection. Overall, the data suggest MVL and CV-N have the potential for toxicity due to interactions with cellular proteins while GNA may be a better therapeutic agent due to specificity for the HCV gpE1E2.

Antibodies to an interfering epitope in hepatitis C Virus E2 can mask vaccine-induced neutralizing activity.

Hepatitis C virus (HCV) neutralization occurring at the E2 region 412‐426 (EP‐I) could be enhanced when antibodies directed specifically to the E2 region 434‐446 (EP‐II) were removed from serum samples of persistently infected patients and vaccinated chimpanzees, a phenomenon of so‐called antibody interference. Here, we show that this type of interference can be observed in individuals after immunization with recombinant E1E2 proteins. One hundred twelve blinded serum samples from a phase I, placebo‐controlled, dose escalation trial using recombinant HCV E1E2 with MF59C.1 adjuvant in healthy HCV‐negative adults were tested in enzyme‐linked immunosorbent assay for binding reactivity to peptides representing the E2 regions 412‐426 (EP‐I) and 434‐446 (EP‐II). All samples were subsequently tested for neutralizing activity using cell‐culture HCV 1a(H77)/2a chimera, HCV pseudotype particles (HCVpp) H77, and HCVpp HCV‐1 after treatment to remove EP‐II‐specific antibodies or mock treatment with a control peptide. Among the 112 serum samples, we found 22 double positive (EP‐I and EP‐II), 6 EP‐II positive only, 14 EP‐I positive only, and 70 double negative. Depleting EP‐II antibodies from double‐positive serum samples increased 50% inhibitory dose (ID50) neutralizing antibody titers (up to 4.9‐fold) in up to 72% of samples (P ≤ 0.0005), contrasting with ID50 neutralization titer increases in 2 of 70 double‐negative samples (2.9%; P > 0.5). In addition, EP‐I‐specific antibody levels in serum samples showed a significant correlation with ID50 neutralization titers when EP‐II antibodies were removed (P < 0.0003). Conclusion: These data show that antibodies to the region 434‐446 are induced during immunization of individuals with recombinant E1E2 proteins, and that these antibodies can mask effective neutralizing activity from EP‐I‐specific antibodies. Elicitation of EP‐II‐specific antibodies with interfering capacity should be avoided in producing an effective cross‐neutralizing vaccine aimed at the HCV envelope proteins.(Hepatology 2015;62:1670–1682)

Modeling of patient virus titers suggests that availability of a vaccine could reduce hepatitis C virus transmission among injecting drug users

A future vaccine could reduce hepatitis C virus transmission among those sharing syringes, even in the presence of postexposure viral replication in vaccinees. Hampering hepatitis C virus transmission No hepatitis C virus (HCV) vaccine is currently available, and evidence from studies in nonhuman primates suggests that any future human HCV vaccine would be unlikely to induce complete immunity against the virus. Major et al. examined whether lowered HCV titers potentially resulting from an imperfect vaccine might still stem HCV transmission in people who inject drugs. The authors measured the HCV RNA from infected human plasma retained in contaminated needles and syringes. Their mathematical model combining these measurements with published HCV viral kinetics data suggested that a partially effective vaccine could reduce the HCV transmission risk among individuals who share contaminated needles and syringes. The major route of hepatitis C virus (HCV) transmission in the United States is injection drug use. We hypothesized that if an HCV vaccine were available, vaccination could affect HCV transmission among people who inject drugs by reducing HCV titers after viral exposure without necessarily achieving sterilizing immunity. To investigate this possibility, we developed a mathematical model to determine transmission probabilities relative to the HCV RNA titers of needle/syringe-sharing donors. We simulated sharing of two types of syringes fitted with needles that retain either large or small amounts of fluid after expulsion. Using previously published viral kinetics data from both naïve subjects infected with HCV and reinfected individuals who had previously cleared an HCV infection, we estimated transmission risk between pairs of serodiscordant injecting drug users, accounting for syringe type, rinsing, and sharing frequency. We calculated that the risk of HCV transmission through syringe sharing increased ~10-fold as viral titers (log10 IU/ml) increased ~25-fold. Cumulative analyses showed that, assuming sharing episodes every 7 days, the mean transmission risk over the first 6 months was >90% between two people sharing syringes when one had an HCV RNA titer >5 log10 IU/ml. For those with preexisting immunity that rapidly controlled HCV, the cumulative risk decreased to 1 to 25% depending on HCV titer and syringe type. Our modeling approach demonstrates that, even with transient viral replication after exposure during injection drug use, HCV transmission among people sharing syringes could be reduced through vaccination if an HCV vaccine were available.

Qualitative differences in cellular immunogenicity elicited by hepatitis C virus T-Cell vaccines employing prime-boost regimens

T-cell based vaccines have been considered as attractive candidates for prevention of hepatitis C virus (HCV) infections. In this study we compared the magnitude and phenotypic characteristics of CD8+ T-cells induced by three commonly used viral vectors, Adenovirus-5 (Ad5), Vaccinia virus (VV) and Modified Vaccinia Ankara (MVA) expressing the HCV NS3/4A protein. C57/BL6 mice were primed with DNA expressing NS3/4A and boosted with each of the viral vectors in individual groups of mice. We then tracked the vaccine-induced CD8+ T-cell responses using pentamer binding and cytokine production analysis. Overall, our data indicate that the memory cells induced by Ad5 were inferior to those induced by VV or MVA. We found that Ad5 boosting resulted in rapid expansion and significantly higher frequencies of NS3-specific T-cells compared to VV and MVA boosting. However, the functional profiles, assessed through analysis of the memory cell marker CD127 and the anti-apoptotic molecule Bcl-2 in the blood, spleen, and liver; and measurements of interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 production indicated significantly lower frequencies of long-lived memory T-cells following Ad5 boosting compared to VV and MVA. This same set of analyses suggested that the memory cells induced following boosting with MVA were superior to those induced by both Ad5 and VV. This superiority of the MVA-induced CD8+ T-cells was confirmed following surrogate challenge of mice with a recombinant mouse herpes virus expressing the HCV NS3 protein. Higher levels of NS3-specific CD8+ T-cells displaying the functional markers CD69, Ki67 and Granzyme B were found in the spleens of mice boosted with MVA compared to VV and Ad5, both alone and in combination. These data suggest that MVA may be a more successful viral vector for induction of effective CD8+ T-cell responses against hepatitis C virus.

Determinants in the IgV Domain of human HAVCR1 (TIM-1) are required to enhance Hepatitis C Virus Entry

ABSTRACT Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry, but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. Human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose-dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucinlike region and was abrogated by a mutation of N to A at position 94 (N94A) in the Ig variable (IgV) domain phospholipid-binding pocket, indicating a direct interaction of the HAVCR1 IgV domain with HCV virions. However, knockout of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV, although replacement of the mouse IgV domain with the human HAVCR1 IgV domain restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid-binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment, leading to direct interactions with specific receptors, such as CD81. IMPORTANCE Hepatitis C virus (HCV) enters cells through a multifaceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking and silencing or knockout of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the interaction of HAVCR1 with HCV early during infection enhances entry but is not required for infection support the hypothesis that HAVCR1 facilitates entry by stabilizing or enhancing virus binding to the cell surface membrane and allowing the correct virus-receptor positioning for interaction with the main HCV receptors. Furthermore, our data show that in addition to the phospholipid-binding function of HAVCR1, the enhancement of HCV infection involves other determinants in the IgV domain of HAVCR1. These findings expand the repertoire of molecules that HCV uses for cell entry, adding to the already complex mechanism of HCV infection and pathogenesis.