Rapid development of a DNA vaccine for Zika virus

Abstract

Zika virus (ZIKV) was identified as a cause of congenital disease during the explosive outbreak in the Americas and Caribbean that began in 2015. Because of the ongoing fetal risk from endemic disease and travel-related exposures, a vaccine to prevent viremia in women of childbearing age and their partners is imperative. We found that vaccination with DNA expressing the premembrane and envelope proteins of ZIKV was immunogenic in mice and nonhuman primates, and protection against viremia after ZIKV challenge correlated with serum neutralizing activity. These data not only indicate that DNA vaccination could be a successful approach to protect against ZIKV infection, but also suggest a protective threshold of vaccine-induced neutralizing activity that prevents viremia after acute infection.

Figure 1. ZIKV DNA vaccine design and characterization

(A) Schematic representation of ZIKV genome and ZIKV DNA vaccine constructs VRC5283 and VRC5288. (B) Expression and secretion of ZIKV E was analyzed by Western blot of transfected 293T cell lysates and SVP precipitate pelleted from culture supernatants through a 20% sucrose cushion demonstrating that the VRC5288 construct secretes more particles than VRC5283. (C) Particle-capture ELISA quantifying the secretion of ZIKV SVP from transfected cells. (D) ZIKV subviral particles (SVP) were purified from the culture supernatant of VRC5288-transfected 293-F cells and subjected to negative staining and electron microscopy. SVP are labeled with arrowheads. The VRC8400 empty backbone plasmid vector was used as a control.

Figure 2. ZIKV DNA vaccines elicit robust binding and neutralizing antibodies in nonhuman primates

Rhesus macaques (n=6/group) were either mock immunized with VRC8400 empty backbone expression plasmid or with VRC5283 or VRC5288 vaccine plasmids intramuscularly with the indicated doses and number of vaccinations. (A) Macaque sera were assayed weekly for ZIKV binding antibodies by ELISA. Each line represents the average titer of an individual animal from 1–2 technical duplicates and the dashed line indicates the limit of detection (reciprocal titer of 64). Any measurement below the limit of detection was assigned a value of half the limit of detection for graphing and statistical purposes. (B) The NAb response elicited by vaccination was analyzed using ZIKV reporter virus particles (RVPs). The dilution of sera required for half-maximal inhibition of virus infection (EC₅₀) was estimated by non-linear regression analysis. Lines connect the average EC₅₀ values of 2–5 independent experiments, each performed with duplicate technical replicates, for the individual monkeys in each group at each timepoint. Error bars denote the standard error of mean. The dotted line denotes the limit of confidence for the RVP assay (reciprocal titer of 60). Measurements below the limit of detection were assigned a value of 30. The average binding antibody (C) and NAb (D) responses for each vaccine group are shown. Error bars denote the standard error of the mean.

Figure 3. ZIKV DNA vaccines reduce viremia in ZIKV-challenged rhesus macaques

Eight weeks after the first vaccination, macaques were challenged with 10³ FFU of ZIKV PRVABC59. (A) qPCR of the capsid gene was used to determine the genome copies/ml on days 1–5 and 7 post-challenge. Each line represents an individual animal. (B) Mean viral load after challenge in each group. Error bars represent the standard error of the mean. Dashed line indicates the limit of detection (100 copies/ml). Any value below the limit of detection was assigned a value half the limit of detection for graphing and AUC calculation.

Figure 4. Protection from ZIKV challenge correlates with NAb titers present at challenge

Animals that had detectable viremia post-challenge were analyzed with respect to pre-challenge NAb activity. (A) The reciprocal EC₅₀ NAb titer of each animal is individually plotted to reflect whether infection occurred or not. Lines indicate individual animals. Protected (no detectable viremia) and infected (viremia detectable on two successive days) animals are represented by gray and red lines, respectively. The sole animal that received two 4 mg doses of VRC5288 and was found to have a low level of viremia on days 3 and 7 after challenge is denoted as “breakthrough” (black outlined dots). (B) The probability of infection (Logit) based on the reciprocal EC₅₀ NAb titer is demonstrated and indicates that prevention of viremia would be expected in approximately 70% of animals with NAb titers >1000.