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.

FIG. 1

Transient expression of HTNV G1 and G2. COS cells were transfected with pWRG/HTN-M or a negative control plasmid (pWRG7077) and, after 24 h, radiolabeled cell lysates were prepared for analysis by RIPA. Expression products were immunoprecipitated with a polyclonal mouse hyperimmune ascitic fluid against HTNV (HTN HMAF), a G1-specific MAb (MAb 6D4), or a G2-specific MAb (MAb 23G10). Molecular size markers (M) are shown in the first lane and sizes in kilodaltons are indicated to the left. The position of G1 and G2 are shown at the right.

FIG. 2

DNA vaccination with plasmid expressing HTNV G1 and G2 protects against HTNV infection. The results of two independent experiments are combined in this figure. In the first experiment, one group of hamsters (659 to 666) was vaccinated with pWRG/HTN-M, and a second negative control group (667 to 674) was vaccinated with the vector plasmid, pWRG7077. In the second experiment one group of hamsters (2101 to 2108) was vaccinated with a slightly modified plasmid, pWRG/HTN-M(x), and a second negative control group (2109 to 2116) remained unvaccinated. Three weeks after the final vaccination, prechallenge serum samples were obtained, and the hamsters were challenged with HTNV. Postchallenge serum samples were collected 28 days after challenge. The pre- and postchallenge serum samples were tested for N-specific antibodies by anti-N ELISA and for neutralizing antibodies by PRNT. The pre- and postchallenge endpoint antibody titer for each hamster is shown. For each experiment, the prechallenge homologous PRNT₈₀ titers were sorted from highest to lowest (left to right).

FIG. 3

Cross-protection. Hamsters were vaccinated with the indicated plasmid [pWRG/SEO-M, pWRG/HTN-M(x), or a negative control], and then challenged with the indicated virus. The negative control hamsters in panels A, B, and C were vaccinated with a pWRG7077-based plasmid; the negative control hamsters in panels D and E remained unvaccinated. Pre- and postchallenge serum samples were tested for anti-N antibodies by anti-N ELISA and for neutralizing antibodies by PRNT. PRNT₈₀ titers for homologous virus and PRNT₅₀ titers for heterologous virus were determined. The prechallenge (■) and postchallenge () endpoint antibody titers for each hamster are shown. Prechallenge homologous PRNT₈₀ titers (sorted from highest to lowest, left to right) are shown as lines with symbols (●). The identification code for each hamster is shown on the x axis. The HTNV PRNT and anti-N ELISA data for hamsters 943, 944, 945, and 948 were published previously (11).

FIG. 4

DNA vaccination with plasmid expressing SEOV or HTNV G1 and G2 elicits high-titer neutralizing antibody responses in rhesus monkeys. Rhesus monkeys were vaccinated with either pWRG/SEO-M(x), pWRG/HTN-M(x), negative control DNA, or rVV/HTN-M+S by the indicated route as described in Materials and Methods. Serum samples were obtained before vaccination (column P) and then 3 weeks after the first (column 1), second (column 2), and third (column 3) vaccinations. The PRNT titer represents the reciprocal serum dilution that reduced virus plaque number by 80% or 50%. The live virus vaccinia recombinant vaccine (rVV/HTN-M+S) was administered only two times at a 6- week interval. The identification code for each monkey is shown below its respective plot.

FIG. 5

Neutralizing antibodies elicited by DNA vaccination are still detected in rhesus monkeys 8 months after the final vaccination. Rhesus monkeys vaccinated with the indicated vaccine were bled 3 weeks after each vaccination and then at 2, 4, 6, and 8 months after the final vaccination (weeks 14, 22, 30, and 38, respectively). The homologous neutralizing antibody response for the indicated week after the first vaccination (week 0) was evaluated by PRNT. Each line represents an individual monkey. The week 9 data are also presented in Fig. 4.