James H. Mathews

Antibody prophylaxis and therapy for flavivirus encephalitis infections.

Abstract: The outbreak of West Nile (WN) encephalitis in the United States has rekindled interest in developing direct methods for prevention and control of human flaviviral infections. Although equine WN vaccines are currently being developed, a WN vaccine for humans is years away. There is also no specific therapeutic agent for flaviviral infections. The incidence of human WN virus infection is very low, which makes it difficult to target the human populations in need of vaccination and to assess the vaccines economic feasibility. It has been shown, however, that prophylactic application of antiflaviviral antibody can protect mice from subsequent virus challenge. This model of antibody prophylaxis using murine monoclonal antibodies (MAbs) has been used to determine the timing of antibody application and specificity of applied antibody necessary for successful prophylaxis. The major flaviviral antigen is the envelope (E) glycoprotein that binds cellular receptors, mediates cell membrane fusion, and contains an array of epitopes that elicit virus‐neutralizing and nonneutralizing antibodies. The protective efficacy of an E‐glycoprotein‐specific MAb is directly related to its ability to neutralize virus infectivity. The window for successful application of prophylactic antibody to prevent flaviviral encephalitis closes at about 4 to 6 days postinfection concomitant with viral invasion of the brain. Using murine MAbs to modify human disease results in a human antimouse antibody (HAMA) response that eventually limits the effectiveness of subsequent murine antibody applications. To reduce the HAMA response and make these MAbs more generally useful for humans, murine MAbs can be “humanized” or human MAbs with analogous reactivities can be developed. Antiflaviviral human or humanized MAbs might be practical and cost‐effective reagents for preventing or modifying flaviviral diseases.

Cellular and humoral immune response to group B streptococci.

Cellular and humoral responses to group B streptococcal challenge were studied in primate plasma. Three components are associated with clinical presentations observed in neonatal sepsis. They are B I a plasma factor, phagocytic activity, and type-specific agglutinins. The usually fatal acute sepsis due to B I a organisms is apparently due to absence of the immune components in most newborn infants. The development of delayed sepsis and meningitis due to type B III streptococci is related to an interaction of challenge dose and individual ability to phagocytize B III cells.

Molecular and biological characterization of a non-glycosylated isolate of St Louis encephalitis virus.

The glycosylation patterns of the envelope (E) glycoprotein of several naturally occurring strains of St Louis encephalitis (SLE) virus were investigated. SLE viruses were found that contained both glycosylated and non-glycosylated E proteins, and one isolate (Tr 9464) that lacks N-linked glycosylation sites on its E protein was identified. SLE virus monoclonal antibodies that define E protein B cell epitopes and demonstrate biological activities reacted essentially to the same extent with glycosylated and non-glycosylated virions. These results indicate that glycosylation is not essential for epitope conformation or recognition. However, failure to glycosylate the E protein was associated with possible morphogenetic differences as manifested by reduced virus yields and differences in specific infectivity.

Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins

cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.

Specificity of the murine T helper cell immune response to various alphaviruses

We investigated the specificity of the T helper (Th) cell immune response to three alphaviruses: Venezuelan equine encephalomyelitis (VEE), eastern equine encephalitis (EEE) and western equine encephalitis (WEE). Single cell suspensions were prepared from spleens of virus-primed C3H mice, and T lymphocyte populations were enriched by nylon wool chromatography. T cells were incubated in vitro with irradiated, syngeneic splenic stimulator cells previously exposed to purified virus. Cellular proliferation was measured by [3H]thymidine uptake 5 days post-stimulation. The predominant proliferating cell type secreted interleukin-2 and was of the Th cell phenotype Thy-1+, Lyt-1+,2-, L3T4+. Stimulation of VEE, EEE and WEE virus-primed Th cells with homologous and heterologous virus resulted primarily in a proliferative response specific for the immunizing virus. The corresponding antibody response, as measured by ELISA using purified virus as antigen, was also specific for the immunizing virus. The magnitude of the blastogenic response of VEE TC-83 virus-primed lymphocytes to a battery of VEE subtype viruses was remarkably similar to schemes of antigenic classification. The results indicate that the dominant Th cell epitopes on these alphaviruses represent regions largely virus-specific and lead to a virus-specific B cell response which does not change over time after primary inoculations of mice with VEE and WEE viruses and multiple inoculations of mice with EEE virus.

Murine T-helper cell immune response to recombinant vaccinia—Venezuelan equine encephalitis virus

The T-helper (Th) cell immune response following immunization of C3H (H-2k) mice with a recombinant vaccinia (VAC) virus (TC-5A) expressing the structural proteins (capsid, E1 and E2) of the attenuated vaccine strain (TC-83) of Venezuelan equine encephalitis (VEE) virus was compared with the immune response induced in mice after immunization with TC-83 virus. TC-5A virus elicited Th cells that strongly recognized both VAC and TC-83 viruses in in vitro lymphoblastogenesis tests. Th-cell activation was associated with elevated levels of interleukin-2. TC-5A virus induced long-term humoral immunity; VEE virus-binding and neutralizing antibodies were detected in mouse sera collected from mice 16 months after a single immunization.

Glycoproteins of Venezuelan Equine Encephalitis (VEE) Virus: Molecular Structure and Function in Virus Pathogenicity and Host Immunity

The VEE virus particle contains a ssRNA genome enclosed in an icosahedral capsid and an envelope containing two glycoproteins, E2 (56k daltons, gpF6) and El (50k daltons, gp50). The amino terminal sequences of the envelope proteins have been determined and compared with data for other alphaviruses. The alphavirus glycoproteins share regions of homology, particularly in the conservation of proline and cysteine residues, implying that the three dimensional conformation is conserved. Deletions and amino acid replacements can occur at most locations with the alphavirus glycoproteins indicating that variation within the proteins can be accommodated without destroying biological function. Tryptic map analysis of the two virus glycoproteins shows differing degrees of diversity within the VEE complex, the larger protein being the more variable. Viral neutralization (N) and hemagglutination inhibition (HAI) determinants have been placed on the gp56 VEE/TC-83 protein using monoclonal antibody to purified proteins. Antigenic analysis indicates the presence of at least three antigenic epitopes on gp56 and four on gp50. Biological functions of N and HAI are primarily associated with gp56°, the critical N site. In vivo protection from virus infection is provided by passive immunization with antisera to gp56 and gp50 as well as highly avid monoclonal antibodies to gp56c, gp50b, gp50c, and gp50d. Small amounts c of gp56c antibodies were needed to provide protection, whereas less avid antibodies to other determinants on gp56 and gp50 were less protective. Protection from VEE virus infection appears to be determined by the specificity of antibody to the virion glycoprotein epitope, avidity of the antibody and the ability of the antibody to react with antigenic epitopes proximal to the critical N site.

Biochemical and Biological Analysis of Venezuelan Equine Encephalomyelitis Viruses (VEE) Using Monoclonal Antibodies

Monoclonal antibodies directed against the surface glycoproteins of TC-83 virus were used to analyze the antigenic structure expressed on the virion surface. Three epitopes were identified on the gp56 and four epitopes were identified on the gp50 using a cross-reactivity assay with closely related VEE viruses and an ELISA competitive binding assay (CBA). Only antibodies directed against the gp56 epitope effectively blocked viral hemagglutination and infectivity. Anti-gp50 antibodies had very low levels of in vitro biologic activity. This activity was Drobably due to the close spatial arrangment this epitope has with the gp56 epitope as demonstrated in the CBA. Antibodies directed against each epitope were used as a source for passive immunization of 3-week-old mice. Protection against subsequent VEE infection could be correlated to both epitope specificity and the ability, to fix complement. The most protective epitopes were the gp56c and gp50b. As little as 5 μg of antibody administered i.v. could protect animals. Those animals that survived challenge demonstrated good anti-viral antibody when tested 25 days post-infection. Viremia studies indicated that the virus was replicating in the spleen, however little virus could be detected in the brain. Antibody fragment studies indicated that the Fc portion was important for in vivo protection.