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. 2004 May;78(9):4433-43.
doi: 10.1128/jvi.78.9.4433-4443.2004.

Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox

J W Hooper  1 , E Thompson, C Wilhelmsen, M Zimmerman, M Ait Ichou, S E Steffen, C S Schmaljohn, A L Schmaljohn, P B Jahrling
Affiliations

Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox

J W Hooper et al. J Virol. 2004 May.

Abstract

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.

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Figures

FIG. 1.
FIG. 1.
Memory antibody response to vaccination with poxvirus DNA vaccines. (a) Sera from rhesus macaques previously (1 to 2 years) vaccinated with the 4pox DNA vaccine, L1R DNA vaccine, negative control (Neg. cont.) DNA vaccine, or Dryvax collected immediately before (pre) and 11 days after (post) a booster vaccination were evaluated for anti-VACV antibodies by ELISA using VACV-infected-cell lysate antigen and VACV PRNT. ELISA end point and PRNT50 titers are shown. *, ELISA titer of <100 or PRNT titer of <20. na, not applicable because there was no boost. (b) Pre- and postboost sera from three monkeys (L201-1, RC114, and CH93) vaccinated with the 4pox DNA vaccine were tested for immunogen-specific antibodies by RIPA using lysates from COS cells transfected with plasmids expressing the four VACV genes that comprise the 4pox DNA vaccine: pWRG/L1R, pWRG/A33R, pWRG/B5R, and pWRG/A27L. Lysate from COS cells transfected with empty-vector plasmid (pWRG7077) was used as a negative control antigen (−). (c) Pre- and postboost sera from two monkeys vaccinated with the L1R DNA vaccine (CH63 and CH74) were tested for L1R-specific antibodies by RIPA using lysates from COS cells transfected with a plasmid, pMPOX/L1Ro, expressing the MPOV L1R orthologous protein, L1Ro (13). Serum from a monkey (CH02) vaccinated with a negative control plasmid and serum from a monkey (CH63) vaccinated with pWRG/L1R collected after the initial series of vaccinations (9) were used as negative (−) and positive (+) controls, respectively. Lysate from COS cells transfected with the empty vector pWRG7077 served as a negative control antigen. Molecular mass markers (M) are shown in kilodaltons on the left, and the positions of immunoprecipitated VACV proteins are shown on the right.
FIG. 2.
FIG. 2.
Evolution of disease in rhesus macaques challenged i.v. with MPOV. (a) Monkeys vaccinated with negative control DNA vaccines were injected (day 0) with a high or low dose of MPOV-Z79. Graphic representations of rash distribution, lesion number, fever, elevated white blood cell counts, and bleeding disorders over 3 weeks are shown. (b) Monkeys vaccinated with a negative control DNA vaccine, 4pox DNA vaccine, L1R DNA vaccine, or Dryvax were injected (day 0) with 2 × 107 PFU of MPOV-Z79. Throat swab and blood viremia data are included in panel b but not in panel a. (c) Umbilicated lesions on face (monkey CH32; day 10 after challenge) 6 days after onset of rash. (d) Deep pustular lesions on CH74 palm (day 10). (e) Disproportionate number of lesions on leg injected with virus (monkey CH63; day 14). (f) Bleeding lesions on CH74 palm (day 14) 8 days after onset of rash.
FIG. 3.
FIG. 3.
Abnormal hematological findings in immunized and control monkeys challenged with MPOV. (a and b) Automated cell counts of whole blood were determined; white blood cell (WBC) counts (a) and hematocrit (b) are shown. (c) Serum clinical chemistries. Monkeys were challenged on day 0 (bold vertical line). The dashed lines indicate normal high and low values for rhesus macaques. †, fatality.
FIG. 4.
FIG. 4.
Time courses of viremia in vaccinated and control monkeys challenged with MPOV. (a) Infectious MPOV detected in throat swabs. (b) Infectious virus detected in whole blood by plaque assay. (c) MPOV genomes detected in whole blood by TaqMan PCR. Monkeys were challenged with MPOV on day 0. †, fatality.
FIG. 5.
FIG. 5.
Antibody responses before and after MPOV challenge. (a) Sera from monkeys vaccinated with 4pox DNA vaccine, L1R DNA vaccine, negative control (Neg. cont.) DNA vaccine, or Dryvax were collected 1 week before MPOV challenge, at the time of challenge (day 0), and at the indicated times after challenge. PRNT 80% neutralization titers are shown. (b) Immunogen-specific antibody titers in sera collected during the week before challenge were determined by ELISA using partially purified A27L, L1R, B5R, or A33R protein expressed in E. coli. Each bar represents the geometric mean titer obtained from at least two experiments. (c) MPOV-specific neutralizing antibody titers in sera collected at the time of MPOV challenge (day 0). The bars represent the mean value of two to four determinations ± standard deviation.
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