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 10 pfu of vsvg / 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 ( vsvg / andvgpc ) ( 5 ) . at 28 days postvaccination
, the 4 nhps were challenged with a lethal dose of lasv ( 10 50% tissue culture infectious doses [ tcid50s ] ) ( 9 ) .
the control animal showed signs of lassa fever 710 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 57 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 vsvg / 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 ) .
* vsv , vesicular stomatitis virus ; vhf , viral hemorrhagic fever ; lasv , lassa virus ; gpc , glycoprotein precursor ; np , nucleocapsid protein ; ebov , ebola virus ; gp , glycoprotein ; vp40 viral protein 40 ; nd , not determined .
titers are indicated as reciprocal endpoint dilutions from elisas for recombinant antigens ( lasv np , ebov gp , and vp40 ) , or whole virus preparations ( vsv ) .
approximately 90 days after the original vaccination with vsvg / lasvgpc , the 3 nhps were vaccinated with a single dose of 10 pfu of vsvg / 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 ( 10 pfu ) ( 10 ) .
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 36 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 1,6006,400 ) 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 vsvg / 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 ) .
results of this study demonstrate that multiple vsv vaccines can be used in a population without any deleterious effect on overall protective efficacy .
 we demonstrated that previous vaccination with a vesicular stomatitis virus ( vsv)based lassa virus vaccine does not alter protective efficacy of subsequent vaccination with a vsv - based ebola virus vaccine . 
 these findings demonstrate the utility of vsv - based vaccines against divergent viral pathogens , even when preexisting immunity to the vaccine vector is present . 
The Study
Conclusions