Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques

Abstract

Background: Zika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV.

Methodology/principal findings: Using our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form.

Conclusions/significance: An effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains.

Fig 1. East African ZIKV MR766 envelope sequences often contain an in-frame deletion of an N-linked glycosylation site and are heterologous with respect to Asian ZIKV.

The amino acid sequences of the Envelope protein for six ZIKV MR766 Genbank sequences were aligned to the consensus amino acid sequences of the three ZIKV MR766 stock viruses (Chal Stck, CDC Stock, and WRCEVA stock) using a Muscle alignment in Geneious. Dots represent identity to the consensus sequence. Dashes represent deletions. Only sections of the E protein with variations are shown, all other parts of the E protein showed no variation. Capital letters represent amino acids. The frequencies of the deletion and the restored deletion are shown below each of the stock sequences. Genbank reference sequence AY632535 had two amino acids that were different from the other reference sequences. The frequency of reads with these amino acid variants as determined by deep sequencing are shown below each of the stock sequences. A. Envelope protein amino acid region 136–178. The gray ellipse above the sequences represent the 150 loop of the E protein [20]. B. Envelope protein amino acid region 271–313.

Fig 2. ZIKV-002 macaques challenged with ZIKV MR766 are protected from heterologous reinfection with ZIKV-FP.

A. Study timeline with dates of primary and secondary, heterologous ZIKV challenges. Samples were collected daily from 0 to 10 dpi, and then weekly thereafter until secondary challenge (denoted by ticks along the timeline). Challenge stocks were derived from the East African and French Polynesian virus strains detailed above the timeline. B. Plasma viral loads, shown as vRNA copies/mL for each of the macaques challenged with 1 x 10⁶ (solid green line), 1x 10⁵ (solid orange line), or 1 x 10⁴ (solid blue line) PFU/mL of ZIKV MR766 challenge stock from the date of primary challenge through 10 days post heterologous challenge with ZIKV-FP. For comparison of plasma viral loads between ZIKV strains, solid light grey lines depict the plasma viral load trajectories for animals that were challenged with the same dose of ZIKV-FP and then rechallenged with homologous ZIKV-FP [22]. C. Oral swab and D. pan urine viral loads.

Fig 3. An N-linked glycosylation site in envelope is rapidly selected in vivo.

Envelope sequences from the three animals were sequenced at three days post infection, and from two of the animals at day six post infection. A Muscle alignment of the translated sequences was generated in Geneious. Dots represent identity to the consensus sequence. Dashes represent deletions. Capital letters represent amino acids. Only regions of the E protein with sequence variants are depicted. A. E protein amino acid positions 136–178. The frequencies of the deletion and the restored deletion are shown below each of the stock sequences, with the indicated site boxed. Amino acid variant frequencies matching the variant sites in Fig 1A are shown. The gray ellipse above the sequence alignment represents the 150 loop of the E protein [20]. B. E protein amino acid positions 271–313. C. E protein amino acid positions 361–450. There were two additional nonsynonymous variants at greater than 5% in animal 562876 at day three, and the frequency of the amino acid variants from the other animals and time points are shown below each sample.

Fig 4. East African ZIKV MR766 infection elicits a robust, multifaceted immune response.

Expansion of Ki-67+ (activated) A. CD8+ T cells (effector memory, central memory or naive) and B. CD4+ T cells (effector memory, central memory or naive) was measured at days 0, 2, 3, and days 7 through 10, then on days 14, 21 and 28 post-infection. After a rest period, activated immune responses were measured at days 63 and 67 post-infection. At 70 dpi, animals were heterologously re-challenged with ZIKV-FP. Expansion of activated cells was measured daily through 80 dpi, then at days 85 and 91 post-infection. Each population is presented as a percentage of the total CD8+ or CD4+ T cell population. C. Total number of plasmablast cells found in PBMCs collected at 3, 7, 10 and 14 dpi for each animal. D. PRNT₉₀ titers 21 days post infection, 63 days post infection, and seven days post rechallenge for ZIKV-002 animals against ZIKV-FP. All three animals did not have detectable nAb titers prior to initial infection.