Connie S. Schmaljohn

Active and Passive Vaccination against Hantavirus Pulmonary Syndrome with Andes Virus M Genome Segment-Based DNA Vaccine

ABSTRACT Hantavirus pulmonary syndrome (HPS) is a rapidly progressing human disease with one of the highest case fatality rates (30 to 50%) of any acute viral disease known. There are no vaccines, effective antiviral drugs, or immunologics to prevent or treat HPS. In an attempt to develop HPS medical countermeasures, we constructed an expression plasmid, pWRG/AND-M, that contains the full-length M genome segment of Andes virus (ANDV), a South American hantavirus. Transfection experiments in cell culture indicated that both the G1 and G2 glycoproteins are expressed from pWRG/AND-M. Rhesus macaques vaccinated by gene gun with pWRG/AND-M developed remarkably high levels of neutralizing antibodies that not only neutralized ANDV but also cross-neutralized other HPS-associated hantaviruses, including Sin Nombre virus. To determine if the antibodies elicited in the monkeys could confer protection, we performed a series of passive-transfer experiments using a recently described lethal HPS animal model (i.e., adult Syrian hamsters develop HPS and die within 10 to 15 days after challenge with ANDV). When injected into hamsters 1 day before challenge, sera from the vaccinated monkeys either provided sterile protection or delayed the onset of HPS and death. When injected on day 4 or 5 after challenge, the monkey sera protected 100% of the hamsters from lethal disease. These data provide a proof of concept for a gene-based HPS vaccine and also demonstrate the potential value of a postexposure immunoprophylactic to treat individuals after exposure, or potential exposure, to these highly lethal hantaviruses.

DNA Vaccination with the Hantaan Virus M Gene Protects Hamsters against Three of Four HFRS Hantaviruses and Elicits a High-Titer Neutralizing Antibody Response in Rhesus Monkeys

ABSTRACT Four hantaviruses—Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus—are known to cause hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. HTNV causes the most severe form of HFRS (5 to 15% case-fatality rate) and afflicts tens of thousands of people annually. Previously, we demonstrated that DNA vaccination with a plasmid expressing the SEOV M gene elicited neutralizing antibodies and protected hamsters against infection with SEOV and HTNV. Here, we report the construction and evaluation of a DNA vaccine that expresses the HTNV M gene products, G1 and G2. DNA vaccination of hamsters with the HTNV M gene conferred sterile protection against infection with HTNV, SEOV, and DOBV. DNA vaccination of rhesus monkeys with either the SEOV or HTNV M gene elicited high levels of neutralizing antibodies. These are the first immunogenicity data for hantavirus DNA vaccines in nonhuman primates. Because a neutralizing antibody response is considered a surrogate marker for protective immunity in humans, our protection data in hamsters combined with the immunogenicity data in monkeys suggest that hantavirus M gene-based DNA vaccines could protect humans against the most severe forms of HFRS.

Diagnostic potential of a baculovirus-expressed nucleocapsid protein for hantaviruses

A non-infectious recombinant, baculovirus-expressed protein, analogous to the nucleocapsid of Hantaan (HTN) virus, and standard Vero E-6 cell culture-prepared HTN antigen, were evaluated by enzyme-linked immunosorbent assays (ELISA) to detect IgG antibodies to various strains of hantaviruses. Rat and human sera previously found to have hantavirusspecific antibodies detectable by the plaque-reduction neutralization test (PRNT) were examined. Results obtained by the immunofluorescent antibody (IFA) test, ELISA, and PRNT were compared. Sera with IFA titers greater than 128 showed a high degree of correlation by both ELISA and PRNT. ELISA tests utilizing baculovirus-expressed protein were almost as sensitive as PRNT in detecting antibodies to HTN, Seoul, and Porogia viruses. The expressed nucleocapsid protein was found to be less sensitive in detecting antibody to Puumala virus, as has been observed with other HTN viral antigen-based tests. Our results suggest that the IgG ELISA with the baculovirus-expressed recombinant nucleocapsid protein is a useful alternative to the IFA for routine screening for antibodies to HTN virus. This test is sensitive, specific, easy to perform, rapid, and requires very small quantities of reagents. Furthermore, the recombinant antigen is non-infectious for humans and is, therefore, safe to prepare and use.