Role of complement and the Fc portion of immunoglobulin G in immunity to Venezuelan equine encephalomyelitis virus infection with glycoprotein-specific monoclonal antibodies.

We have previously characterized with monoclonal antibodies (MAbs) seven unique epitopes on the two envelope glycoproteins of Venezuelan equine encephalomyelitis (VEE) virus vaccine strain TC-83. The epitopes important in protection from VEE virus infection were determined in passive antibody transfer studies, with virulent VEE (Trinidad donkey) virus as the challenge virus. Selected high-avidity MAbs to the three major protective epitopes (E2c, E1b, and E1d) were assayed for in vitro complement activity. All three fixed murine complement to high titer. Limited pepsin digestion of the anti-E2c in the presence of cysteine resulted in a rapid decrease and complete loss of complement-fixing ability by 2 h, but the majority of mice, except at the lowest dilution of MAb, were protected until the Fc termini were cleaved at 3 h. Anti-E2c F(ab')2 would neutralize VEE (Trinidad donkey) virus more efficiently than either Fab' or Fab; none of the fragments would fix complement or was effective in passive protection. C5-deficient mice and mice depleted of C3 with cobra venom factor were still protected from VEE (Trinidad donkey) virus challenge after passive transfer of either anti-E2c or anti-E1b MAb. The results show that the anti-E2c MAb mediates neutralization through bivalent binding at a critical site on the virion and that Fc effector functions, other than complement, are necessary for protection. Although the ability of the anti-E2c MAb to fix complement was associated with its ability to protect in vivo, no direct cause-and-effect relationship was found. Since the epitope defined by the anti-E1d antibody is found on the cell membrane, but is not expressed on the infectious virion, protection in mice was most likely mediated at the cellular level, possibly by inhibition of the final stages of virion maturation.     

Monoclonal antibody cure and prophylaxis of lethal Sindbis virus encephalitis in mice

Neuroadapted Sindbis virus (NSV) causes acute encephalitis and paralyzes and kills adult mice unless they are treated with primary immune serum after infection. To study the nature and specificity of curative antibodies, we gave mice 30 different monoclonal antibodies (MAbs) against Sindbis virus (SV) 24 h after lethal intracerebral inoculation of NSV. By the time of MAb treatment, NSV replication in the brain had been well established (7.5 X 10(7) PFU/g). Seventeen MAbs directed against multiple biological domains on the NSV E1 and E2 envelope glycoproteins prevented paralysis and death. Anticapsid MAbs failed to protect. Altogether, 15 of 17 curative MAbs either neutralized NSV infectivity or lysed NSV-infected cells with complement, but neither ability was necessary or sufficient to guarantee recovery. All 5 protective anti-E2 MAbs neutralized NSV infectivity; 6 of 10 protective anti-E1 MAbs neutralized NSV; 4 did not. Plaque assay or immunohistochemical staining showed that neutralizing and nonneutralizing curative MAbs decreased NSV in the brain, brainstem, and spinal cord. Despite high neutralization titers, hyperimmune anti-SV and anti-NSV mouse sera prevented only 6 and 30% of deaths, respectively, while primary immune sera prevented 50 (SV) and 90% (NSV) of deaths. Secondary intravenous immunization with a live virus apparently diminished, obscured, or failed to boost a class of protective antibodies. When separate mouse groups were given these 30 MAbs 24 h before lethal intracerebral inoculation of NSV, a slightly different set of 17 neutralizing or nonneutralizing anti-E1 and anti-E2 antibodies protected. Two nonneutralizing MAbs and hyperimmune anti-SV serum, which had failed to promote recovery, prophylactically protected 100% of the mice. The antibody requirements or mechanisms of prophylaxis and recovery may differ.     

Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II

The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.     

Properties of monoclonal antibodies against glycoproteins of western equine encephalitis virus.

To analyze the biological activities of the alphavirus glycoproteins, eight different monoclonal antibodies against the two glycoproteins of western equine encephalitis virus were isolated. Five of the eight monoclonal antibodies were shown to be specific for E1 and three for E2 protein by an enzyme-linked immunosorbent assay and by radioimmunoprecipitation. Three of the five anti-E1 and all of the anti-E2 monoclonal antibodies inhibited hemagglutination by purified virions. One anti-E1 and two anti-E2 monoclonal antibodies possessed high virus-neutralizing activity.     

Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.     

Murine Humoral Immune Response against Recombinant Structural Proteins of Hepatitis C Virus Distinct from Those of Patients

We examined the humoral immune response to recombinant structural proteins of hepatitis C virus (HCV) such as C, E1 and E2 in immunized mice. Mice showed high induction of antibodies against these three structural proteins. Conformational and/or linear epitopes of these regions showed high responses in mice. Comparison with patients revealed higher anti-E1 and anti-E2 responses in mice and 15 immunoreactive peptides which are unique to mice, especially 11 peptides from the E2 region. The hydrophilic regions of these proteins were found to be the most immunogenic. Therefore, the murine immune system against recombinant E1 and E2 glycoproteins was distinct from those of patients in natural infection, and may be a target to find protective activity against HCV infection.     

High-Avidity and Potently Neutralizing Cross-Reactive Human Monoclonal Antibodies Derived from Secondary Dengue Virus Infection

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination.     

Sero-Prevalence and Cross-Reactivity of Chikungunya Virus Specific Anti-E2EP3 Antibodies in Arbovirus-Infected Patients

Chikungunya virus (CHIKV) and clinically-related arboviruses cause large epidemics with serious economic and social impact. As clinical symptoms of CHIKV infections are similar to several flavivirus infections, good detection methods to identify CHIKV infection are desired for improved treatment and clinical management. The strength of anti-E2EP3 antibody responses was explored in a longitudinal study on 38 CHIKV-infected patients. We compared their anti-E2EP3 responses with those of patients infected with non-CHIKV alphaviruses, or flaviviruses. E2EP3 cross-reactive samples from patients infected with non-CHIKV viruses were further analyzed with an in vitro CHIKV neutralization assay. CHIKV-specific anti-E2EP3 antibody responses were detected in 72% to 100% of patients. Serum samples from patients infected with other non-CHIKV alphaviruses were cross-reactive to E2EP3. Interestingly, some of these antibodies demonstrated clearly in vitro CHIKV neutralizing activity. Contrastingly, serum samples from flaviviruses-infected patients showed a low level of cross-reactivity against E2EP3. Using CHIKV E2EP3 as a serology marker not only allows early detection of CHIKV specific antibodies, but would also allow the differentiation between CHIKV infections and flavivirus infections with 93% accuracy, thereby allowing precise acute febrile diagnosis and improving clinical management in regions newly suffering from CHIKV outbreaks including the Americas.     

Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses.

Western equine encephalomyelitis (WEE) virus (Togaviridae: Alphavirus) was shown previously to have arisen by recombination between eastern equine encephalomyelitis (EEE)- and Sindbis-like viruses (C. S. Hahn, S. Lustig, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 85:5997-6001, 1988). We have now examined the recombinational history and evolution of all viruses belonging to the WEE antigenic complex, including the Buggy Creek, Fort Morgan, Highlands J, Sindbis, Babanki, Ockelbo, Kyzylagach, Whataroa, and Aura viruses, using nucleotide sequences derived from representative strains. Two regions of the genome were examined: sequences of 477 nucleotides from the C terminus of the E1 envelope glycoprotein gene which in WEE virus was derived from the Sindbis-like virus parent, and 517 nucleotide sequences at the C terminus of the nsP4 gene which in WEE virus was derived from the EEE-like virus parent. Trees based on the E1 region indicated that all members of the WEE virus complex comprise a monophyletic group. Most closely related to WEE viruses are other New World members of the complex: the Highlands J, Buggy Creek, and Fort Morgan viruses. More distantly related WEE complex viruses included the Old World Sindbis, Babanki, Ockelbo, Kyzylagach, and Whataroa viruses, as well as the New World Aura virus. Detailed analyses of 38 strains of WEE virus revealed at least 4 major lineages; two were represented by isolates from Argentina, one was from Brazil, and a fourth contained isolates from many locations in South and North America as well as Cuba. Trees based on the nsP4 gene indicated that all New World WEE complex viruses except Aura virus are recombinants derived from EEE- and Sindbis-like virus ancestors. In contrast, the Old World members of the WEE complex, as well as Aura virus, did not appear to have recombinant genomes. Using an evolutionary rate estimate (2.8 x 10(-4) substitutions per nucleotide per year) obtained from E1-3' sequences of WEE viruses, we estimated that the recombination event occurred in the New World 1,300 to 1,900 years ago. This suggests that the alphaviruses originated in the New World a few thousand years ago.     

Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice

Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species.     

Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein

Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (V(H)) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of V(H) may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The V(H) sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies.     

The gene order for rubella virus structural proteins is NH2-C-E2-E1-COOH

The order of translation in vivo of the genes coding for rubella virus structural proteins was studied in infected B-Vero cells. The proteins were sequentially pulse-chase labeled with [35S]methionine after synchronization of translation initiation with hypertonic salt treatment. A sequential labeling procedure ("window-labeling") to specifically label defined segments of the structural proteins was also used. The labeled proteins were identified by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation with specific antisera directed against the two virion glycoproteins (E1 and E2a/E2b) and the nucleocapsid (C) protein. The order of translation was found to be NH2-C-E2-E1-COOH. We have previously shown that the structural proteins are synthesized in vitro from a cytoplasmic 24S subgenomic mRNA as a 110,000-dalton (p110) precursor (Oker-Blom et al., J. Virol. 49:403-408, 1984). Here, it is shown that p110 is precipitated with anti-C, anti-E2, and anti-E1 sera, indicating that p110 is the precursor of all three structural proteins. Two major in vitro translation products (Mrs, 66,000 and 62,000) that could represent preterminated polypeptide chains or proteolytic cleavage products were precipitated with anti-C and anti-E2 sera, but not with anti-E1 serum, indicating, in conformity with the in vivo results, that the genes for the C and E2 proteins are adjacent to each other. Using these specific antisera, we have also confirmed the identity of the unglycosylated forms of E1 (Mr, 53,000) and E2 (Mr, 30,000) immunoprecipitated from tunicamycin-treated infected cells.     

B cell overexpression of FCRL5 and PD-1 is associated with low antibody titers in HCV infection

Antibodies targeting the hepatitis C virus (HCV) envelope glycoprotein E2 are associated with delayed disease progression, and these antibodies can also facilitate spontaneous clearance of infection in some individuals. However, many infected people demonstrate low titer and delayed anti-E2 antibody responses. Since a goal of HCV vaccine development is induction of high titers of anti-E2 antibodies, it is important to define the mechanisms underlying these suboptimal antibody responses. By staining lymphocytes with a cocktail of soluble E2 (sE2) glycoproteins, we detected HCV E2-specific (sE2+) B cells directly ex vivo at multiple acute infection timepoints in 29 HCV-infected subjects with a wide range of anti-E2 IgG titers, including 17 persistently infected subjects and 12 subjects with spontaneous clearance of infection. We performed multi-dimensional flow cytometric analysis of sE2+ and E2-nonspecific (sE2-) class-switched B cells (csBC). In sE2+ csBC from both persistence and clearance subjects, frequencies of resting memory B cells (rMBC) were reduced, frequencies of activated MBC (actMBC) and tissue-like MBC (tlMBC) were increased, and expression of FCRL5, an IgG receptor, was significantly upregulated. Across all subjects, plasma anti-E2 IgG levels were positively correlated with frequencies of sE2+ rMBC and sE2+ actMBC, while anti-E2 IgG levels were negatively correlated with levels of FCRL5 expression on sE2+ rMBC and PD-1 expression on sE2+ actMBC. Upregulation of FCRL5 on sE2+ rMBC and upregulation of PD-1 on sE2+ actMBC may limit anti-E2 antibody production in vivo. Strategies that limit upregulation of these molecules could potentially generate higher titers of protective antibodies against HCV or other pathogens.     

The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.     

Western equine encephalitis in avian populations in North Dakota, 1975

The involvement of wild birds in western equine encephalitis (WEE) and St. Louis encephalitis (SLE) virus activity in the Red River valley area of North Dakota (USA) during a WEE epidemic was investigated in August 1975. Free-ranging birds were captured with mist nets and nestlings by hand. Virologic and serologic results indicated that a similar rate of WEE virus activity occurred throughout Richland County and between permanent and summer resident birds. The rate of SLE virus activity in the birds of Richland County was lower than for WEE virus, but the SLE antibody prevalence was greater in rural areas than within urban locations. Seven of the nine WEE virus isolations were from nestling birds of four different species; the remaining two from adults of two different species. Overall prevalence of neutralizing (N) antibody against WEE virus was 5% in nestling and 14% in adult birds but was the opposite for N antibody against SLE virus, 17% in nestling and 5% in adult birds. Differences between the two viruses in the presence and persistence of maternal N antibody or differential mortality in nestling birds may have caused the disparity in antibody prevalences.     

Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.     

Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes.     

Fusion to C3d enhances the immunogenicity of the E2 glycoprotein of type 2 bovine viral diarrhea virus

The use of DNA and protein subunit vaccines in animals provides an opportunity to introduce vaccines that are arguably the safest that can be developed. For that reason, considerable effort is under way to devise methods of enhancing the immunogenicity of such vaccines. Seven years ago it was shown that fusing complement fragment C3d to hen egg lysozyme (HEL) enhanced the immunogenicity of HEL 10,000-fold. Based on this observation, we decided to evaluate the effect of C3d on the immunogenicity of the E2 protein of bovine viral diarrhea virus (BVDV). E2 is the major target of neutralizing antibody during BVDV infection. To test the effect of C3d on E2 immunogenicity, expression cassettes encoding a secreted form of E2 alone (E2s) or E2 fused to three copies of murine C3d (E2s-C3d) were constructed. The proteins were purified from the supernatants of transfected cells and used to immunize mice. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for E2s-specific antibody and by a virus neutralization test. The ELISA results indicated that the E2s-C3d protein is 10,000-fold more immunogenic than the E2s protein alone. The maximum primary immune response was elicited with <0.1 microg of E2s-C3d protein without an adjuvant. In addition, we have shown for the first time that high levels of anti-E2s and neutralizing antibodies can be elicited when this same low concentration of E2s-C3d is used to both prime and boost the immune response. We conclude that the E2s-C3d fusion protein has significant potential as a subunit vaccine against BVDV infection.     

Envelope antigens of Sindbis virus in cells infected with temperature-sensitive mutants.

Indirect fluorescent-antibody studies of living and fixed chick cells infected with temperature-sensitive mutants of Sindbis virus suggest that functional envelope glycoprotein E1 must be inserted through the plasma membrane before E2. PE2 and E2 do not affect the insertion of E1. The experiments also suggest that normal PE2, a glycosylated precursor to E2, reacts with anti-E2 serum; the abnormal PE2 made by a temperature-sensitive PE2 cleavage-defective mutant did not. Abnormal E1 proteins made by E1-defective mutants also failed to react with anti-E1 serum.     

Possible Evidence for Interference with Venezuelan Equine Encephalitis Virus Vaccination of Equines by Pre-Existing Antibody to Eastern or Western Equine Encephalitis Virus, or Both

During 1971, an epizootic of Venezuelan equine encephalitis (VEE) reached the United States. Laboratory tests were performed on a large number of sick, healthy, unvaccinated, and vaccinated horses. Neutralization (N) tests in cell cultures revealed that 153 of 193 (79.3%) equines outside the state of Texas and 175 of 204 (85.8%) within Texas (82.6% overall) had detectable N antibody to VEE virus a week or more after vaccination. Twenty-six of 40 (65%) non-Texas equines and 18 of 29 (62%) Texas equines which had no detectable antibody against VEE virus a week or more after vaccination had N antibody against Eastern equine encephalitis (EEE) or Western equine encephalitis (WEE) virus or both, whereas only 50 of 153 (32.7%) non-Texas equines and 82 of 175 (46.9%) Texas equines with demonstrable N antibody against VEE also had N antibody against EEE and/or WEE virus. In vaccinated equines, significant negative correlations were found between the occurrence of antibody to VEE and antibody to EEE and/or WEE virus. These findings support the hypothesis that pre-existing antibody to EEE and/or WEE virus may modify or interfere with infection by VEE virus. The epizoologic significance of this possibility is discussed briefly.