This study was conducted in accordance with a protocol approved by an Institutional Animal Care and Use Committee of the National Institutes of Health. All laboratory work with potentially infectious materials was conducted in a Biosafety Level facility at the Rocky Mountain Laboratories (Division of Intramural Research/National Institute of Allergy and Infectious Diseases/National Institutes of Health).

Three cynomologus macaques were vaccinated with 1 dose of 10⁷ PFU of VSVΔG/LASVGPC, a live-attenuated, recombinant viral vaccine in which the VSV surface glycoprotein has been replaced with those of LASV, by intramuscular injection as described (4). Another age-matched control animal was vaccinated with an irrelevant VSV-based vaccine (VSVΔG/ANDVGPC) (5). At 28 days postvaccination, the 4 NHPs were challenged with a lethal dose of LASV (10⁴ 50% tissue culture infectious doses [TCID₅₀s]) (9). The control animal showed signs of Lassa fever 7–10 days postinoculation and was euthanized 13 days postchallenge because of severity of disease. Classic indicators of Lassa fever, including decreased total protein and albumin; increased serum levels of alanine aminotransferase, aspartate aminotransferase, amylase, blood urea nitrogen, and alkaline phosphatase; and hematologic abnormalities, including thrombocytopenia and lymphopenia, were apparent in this animal.

Virus isolation conducted for select tissue samples showed LASV titers of 5–7 log₁₀ TCID₅₀/g of tissue; blood samples collected on day 10 and at the time of euthanasia (5 and 6.25 log₁₀ TCID₅₀/mL, respectively) showed viremia. In contrast, the 3 animals vaccinated with VSVΔG/LASVGPC resisted lethal LASV challenge and did not demonstrate any clinical signs of disease or any hematologic or biochemical indicators of LASV infection. At no point in the study was virus found in blood samples collected regularly from these 3 animals, even when tested by sensitive reverse transcription PCRs. An ELISA with serum samples collected 45 days postchallenge demonstrated equivocal antibody titers (100) against a recombinant LASV nucleocapsid protein in 1 NHP. The other 2 animals did not show seroconversion, which suggested that vaccination caused nearly sterile immunity against LASV (Table).

Approximately 90 days after the original vaccination with VSVΔG/LASVGPC, the 3 NHPs were vaccinated with a single dose of 10⁷ PFU of VSVΔG/EBOVGP by intramuscular injection as described (3). An additional NHP was vaccinated with a control vaccine as outlined above and served as the inoculation control. At the time of vaccination, the 3 macaques had a robust VSV-specific antibody response with titers of 25,600, as determined by a whole virus ELISA (Table). Despite this finding, the 3 animals that received the VSV-based EBOV vaccine mounted an efficient response to the EBOV glycoprotein (Table) and were completely protected when challenged 28 days later with a lethal dose of EBOV (10³ PFU) (10). Postchallenge, the 3 NHPs did not show any clinical signs of disease. Hematologic and serum biochemistry values remained constant throughout the study, and virus was not found in blood samples collected regularly and tested by using real-time reverse transcription PCR.

In contrast, severe EBOV hemorrhagic fever developed in the control animal, which was characterized by increased serum concentrations of alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase; thrombocytopenia; and viremia (≥7 log₁₀ TCID₅₀/mL whole blood) beginning 3–6 days postchallenge. This animal was euthanized 7 days postchallenge, and titration of selected tissue samples showed EBOV titers of >9 log₁₀ TCID₅₀/g tissue. An ELISA conducted at the conclusion of the study (42 days post–EBOV challenge) showed increased antibody responses to VSV (titers =102,400) and seroconversion to the EBOV viral protein 40 antigen (titers 1,600–6,400) in the 3 surviving NHPs (Table), which is consistent with published results (10).

Because of the remote locations where VHF agents are present, an overall shortage of health care professionals and clinics in these locations, and mobility of human populations, any vaccine against these pathogens would ideally need to elicit a protective immune response after a single vaccination. For this reason, replication-competent viral vectors are considered leading VHF vaccine candidates.

As the VSVΔG/EBOVGP vaccine heads toward clinical trials, it is necessary to clarify the potential limitations of using the VSV platform against multiple VHF agents. A major drawback for many viral vector platforms is preexisting immunity against the vector itself, which can decrease or nullify the essential protective immune response, which results in vaccine failure. Design of the VSV-based vaccines, which encode and express glycoproteins from various pathogens without its own glycoprotein (11,12), suggest that preexisting immunity would not influence protective efficacy of individual vaccinations (12). However, until now, this possibility has not been examined in disease models. Results of this study demonstrate that multiple VSV vaccines can be used in a population without any deleterious effect on overall protective efficacy.