Zika Virus Vaccines

Transmission, Discovery, Global Distribution and Spread

• i.)

  Transmission

Human ZIKV infection is primarily mediated through autochthonous, mosquito-borne transmission by Aedes aegypti mosquitoes, although transmission by other Aedes species may be possible. ^, In utero transmission of ZIKV to the fetus is the cause of congenital ZIKV syndrome (CZS), and likely occurs via a transplacental route by which ZIKV in the maternal blood crosses the placenta to infect the fetus, (reviewed in ) although other modes of maternal transmission may be possible. The persistence of ZIKV in blood, semen, urine, and female genital secretions ^, for extended periods allows for transmission through sexual contact ^, and the transfusion of contaminated blood products may also be a source of contagion. ^,

• ii.)

  Discovery and prepandemic spread

ZIKV was first isolated in 1947 by the Ugandan Virology Research Institute from sentinel monkeys in the Zika Forest. The first isolation of ZIKV from humans occurred 7 years later in Nigeria. Subsequently it was recognized as the cause of sporadic small outbreaks in Africa and Asia. The first notable ZIKV outbreak occurred on the island of Yap in 2007 where an estimated 73% of the population was infected. This was followed by outbreaks in the Pacific Islands of French Polynesia and Tahiti in 2013 and the Cook Islands, Easter Island, and New Caledonia in 2014. Retrospective serological analysis revealed that circulation of ZIKV in Asia, including Bangladesh, Cambodia, Thailand, and the Lao People’s Democratic Republic, occurred prior to and concurrently with these reports. Despite the circulation of ZIKV in Africa and Asia, an association between ZIKV infection and congenital abnormalities or GBS had not been previously appreciated.

• iii.)

  Introduction into the Americas

The first case of ZIKV in the Americas was reported in Brazil in May 2015, although the introduction of the virus into the Western Hemisphere may have been much earlier. For example, ZIKV may have been established years earlier through the arrival of infected participants to athletic events in northern Brazil. Reports of ZIKV infections quickly followed throughout South and Central America, the Caribbean, and parts of North America. The peak of case reporting in the Americas occurred in February 2016 but varied by country and subregion (reviewed in ^, ). By March 2017, over 750,000 suspected and confirmed cases of ZIKV were reported in countries and territories of the Americas. Brazil accounted for almost half (46%) of these cases with Colombia (14%) and Venezuela (8%) being the next largest contributors. South America accounted for the highest number of cases by region (70%), but the Caribbean reported the highest incidence rate (10,510 cases/100,000 population). In the United States of America, most of the approximately 5000 ZIKV cases were associated with travel to regions experiencing ZIKV transmission, including the Caribbean, Central and South America, and other parts of North America. ^, Local transmission was reported in Florida and Texas, and forty-six cases were reported through sexual transmission with partners who had traveled to regions with ZIKV activity. ^, The US territories of Puerto Rico, the US Virgin Islands, and American Samoa had higher incidence rates of ZIKV, totaling 36,432 cases between January 2015 and April 2017, with Puerto Rico accounting for 96.8% of the cases.

• iv.)

  Reintroduction into Africa

Prior to the 2015 epidemic, there is evidence of ZIKV transmission in Burkina Faso, Burundi, Cameroon, Central African Republic, Cote d’Ivoire, Gabon, Nigeria, Senegal, Sierra Leone, and Uganda. ZIKV infections have since been documented in Cabo Verde, Guinea-Bissau, Angola, and retrospectively in Ethiopia. ZIKV-associated microcephaly was identified in 2015-2016 in Cabo Verde, and later in 2017 in Angola. Viruses sequenced from microcephaly cases in Angola and Cabo Verde outbreaks were of the same Asian linage virus identified in the American epidemics, suggesting reintroduction of ZIKV into Africa during the pandemic.

• v.)

  Postpandemic epidemiology and potential for reemergence

While ZIKV transmission has dramatically waned since the peak of the pandemic, its impact on global health was significant. Overall, autochthonous, mosquito-borne ZIKV transmission was documented in 87 countries and territories across Africa, the Americas, South-East Asia, and the Western Pacific (WHO July 2019 Update). Seroprevalence studies in Brazil identified infection rates as high as 73%, although this was not uniform among the populations sampled. ^, Travel-associated ZIKV infections were reported in Europe, but not autochthonous, mosquito-borne transmission. Despite the extensive distribution of ZIKV activity, the spatially heterogeneous attack rates characteristic of the epidemic left many communities vulnerable. Aedes aegypti vectors are established in 61 counties that have not yet reported ZIKV cases and ZIKV cases continue to be reported in regions of the Americas, Asia, and Africa, albeit at a much lower level than the peak in 2016. These circumstances present an opportunity for a resurgence of ZIKV through spread to new regions or as herd immunity wanes in endemic regions.

Classification and Phylogenetics

ZIKV is an enveloped positive-sense, single-stranded RNA virus in the Flavivirus genus of the Flaviviridae family. The flavivirus genus includes YFV, DENV, JEV, West Nile virus (WNV), TBEV, and Powassan virus, among others of significance to public health. Flaviviruses cluster antigenically into serocomplexes. ZIKV is grouped within the Spondweni virus serocomplex and is more closely related to viruses within the DENV and YFV serocomplexes (viruses that are also transmitted by Aedes mosquitoes) than to viruses in the JEV, Nitaya virus, Aroa virus, and Kokobera serocomplexes (which are transmitted by Culex mosquitoes) (reviewed in ). Co-circulation of multiple flaviviruses in regions in Asia and South America coupled with serological cross-reactivity and similar clinical symptoms make it difficult to differentiate ZIKV infection from infections with other flaviviruses and complicate the development of specific diagnostics.

Phylogenetic analysis has defined African and Asian lineages of ZIKV, with the former divided into East African and West African genotypes. ^, The sequence of the envelope (E) protein, a principal target of the humoral response, is highly conserved with ∼96–97% amino acid homology between lineages. By comparison, ZIKV and DENV share roughly 55% of amino acid identity in the E protein. ZIKV constitutes a single serotype and infection with one lineage protects against infection by strains of the other lineage. ^, Recent human outbreaks characterized by a higher frequency of cases of microcephaly and GBS have been attributed to the Asian lineage ZIKV strains, sparking efforts to define genetic differences between Asia and African lineage strains responsible for increased pathogenesis. Multiple mutations have been identified that moderate pathogenesis in animal models and transmissibility. ^, A S139N mutation in the structural protein premembrane protein (prM) has been shown to increase microcephaly in mouse models and was found in outbreaks where higher levels of microcephaly in humans were reported. Other studies suggest that the African lineage ZIKV is more pathogenic and transmissible than Asian lineage viruses in some contexts. ^{, ,}

Genome Structure

The ∼10.7kb RNA genome of ZIKV encodes a single open reading frame (ORF) that is flanked on each end by highly structured untranslated regions (UTR) ( Fig. 65.1A ). A single copy of the genomic RNA is packaged into virions. This molecule has multiple functions that include serving as a substrate for the translation of all viral gene products and as the template for the synthesis of minus-strand RNAs during genome replication. The genomic RNA may also be processed by a cellular 5’-3’ exonuclease to create subgenomic small flavivirus RNAs (sfRNA) that are involved in the regulation of the innate host response to infection. ^,

ZIKV Structural Proteins and Virion Organization

Flaviviruses encode at least ten functional proteins in a single open reading frame that is translated on membranes derived from the endoplasmic reticulum (ER) and subsequently cleaved by host and viral proteases. The three structural proteins (capsid (C), premembrane (prM), and envelope (E)) that comprise the virion are encoded at the 5’ end of the ORF ( Fig. 65.1A ). The C protein is a small (122 amino acid) protein incorporated into virions in complex with the viral RNA genome The capsid protein has numerous non-structural roles in the virus replication cycle that are reviewed elsewhere.

The prM glycoprotein is composed of seven β-strands held together by three disulfide bonds. ^, It is incorporated into virions via connections to the viral membrane mediated by two antiparallel transmembrane domains ( Fig. 65.1B ). ^, prM is cleaved by a host serine furin-like protease during virion transit through the secretory pathway, resulting in the formation of a 75 amino acid membrane (M) peptide that is retained in the virion ( Fig. 65.2 ). ^, The function of the M peptide on virions is unknown.

The principal function of the E protein is to orchestrate virus assembly and cellular entry steps of the replication cycle. High-resolution structures of the E protein have been determined for a soluble E protein ectodomain and when E proteins are organized into virus particles. ^{, ,} The E protein is an elongated three-domain structure connected to the viral membrane by two helical domains (the stem) and two antiparallel transmembrane domains ( Fig. 65.1B ). E protein domains I-III (E-DI, E-DII, and E-DIII) are connected by short flexible linkers that enable motion between these regions during structural transitions that occur during the virion maturation process and low pH-mediated fusion. E-DIII is an immunoglobulin-like fold shown for some flavivirus strains to participate in host cell binding to glycosaminoglycan attachment factors on host cells ; a role for these host molecules during ZIKV entry is not clear.

Flavivirus Nonstructural Proteins

Flaviviruses encode seven nonstructural proteins that play numerous roles in the virus replication and modulation of the host immune response including the antagonism of innate sensing and effector functions, the evasion of complement, and the modulation of inflammasome activity. These proteins and their roles in the viral replication cycle have been reviewed extensively. Of these, nonstructural protein 1 (NS1) is highly immunogenic and has been used as an antigen in several promising flavivirus vaccine candidates. NS1 is a secreted glycoprotein that oligomerizes into dimers that localize to viral replication complexes or the plasma membrane. NS1 hexamers are released from virus-infected cells. For some viruses, NS1 antigen is present at high levels and contributes to pathogenesis via a capacity to activate complement or ability to alter endothelial barrier functions and thereby contribute to viral dissemination.

Virion Morphogenesis and Structure

Flavivirus RNA replication occurs within a network of host membrane compartments induced by the expression of the viral nonstructural proteins. The budding and assembly of flaviviruses are not well understood. Coordination between structural gene expression, proteolytic cleavage of the capsid, and viral RNA replication is suggested by numerous studies. Newly formed virions bud into the lumen of the ER as immature virions. While the structure of these immature virions has been solved for ZIKV and other flaviviruses, ^, the cell biology of the budding event has not been captured at high resolution. Immature virions incorporate 180 copies of the prM and E proteins arranged as heterotrimeric spikes ( Fig. 65.1C ). Sixty of these complexes are arranged on the virion with icosahedral symmetry.

Flavivirus maturation is an essential step in the flavivirus replication cycle defined by the cleavage of prM. Virion maturation occurs in the trans-Golgi network in a pH-dependent manner. Exposure of the immature virion to an acidic environment triggers a reorganization of prM-E trimers into antiparallel E dimers ( Fig. 65.2 ). In this arrangement, prM protrudes through a hole at the dimer interface. This conformation exposes sequence on prM recognized by the furin protease, enabling cleavage and the formation of the M peptide. Subsequent exposure of the virion to a neutral pH of the extracellular space results in the release of the cleaved “pr” protein from the surface and the formation of the mature virion ( Fig. 65.1C ). ^, E proteins of mature virions are arranged in a herringbone pattern of E dimers that lie flat against the viral membrane. ^{, ,}

Virus Particle Heterogeneity

Flaviviruses exist as multiple structures. Three important facets of flavivirus biology have been studied that extend an understanding of what flaviviruses and flavivirus antigens look like under different conditions.

While prM cleavage is required for the formation of infectious virions, it may be inefficient. Tomographic studies of nonuniform DENV revealed a mosaic of regions of E proteins arranged in mature (homodimers) and immature (prM-E heterotrimers) configurations ( Fig. 65.2 ). This heterogeneity markedly impacts antibody recognition as many epitopes are characterized by limited accessibility among the densely arranged E dimers of mature virus. ^, While partially mature virions have been studied extensively using cell lines, what viruses look like in vivo is of critical importance. An analysis of DENV serotype one (DENV1) virions isolated from human plasma revealed cleavage of prM on this population of viruses was very efficient. Important unanswered questions remain and additional studies of different viral strains, viruses isolated from different compartments, and from different hosts are needed.

Virus “breathing” describes the sampling of an ensemble of structural states by virions or capsids at equilibrium and has been observed for enveloped and nonenveloped viruses. ^, Multiple studies identified structures of DENV at physiological temperatures that are markedly different than the herringbone configuration of E proteins characteristic of mature virions. Viral structural dynamics Provide a mechanism to explain antibody recognition of epitopes that are not predicted to be exposed on the surface of mature virions. In support of this concept, time- and temperature-dependent patterns of neutralization have been observed for multiple flaviviruses. ^, Sensitivity to neutralization by antibodies that bind structurally inaccessible epitopes may also be regulated allosterically. ^, This complex biology may have a significant impact on the structures selected for flavivirus immunogens and their capacity to elicit a protective antibody repertoire.