A Vaccine Based on a Modified Vaccinia Virus Ankara Vector Expressing Zika Virus Structural Proteins Controls Zika Virus Replication in Mice
A Vaccine Based on a Modified Vaccinia Virus Ankara Vector Expressing Zika Virus Structural Proteins Controls Zika Virus Replication in Mice
Mar?a Q. Mar?n
Nereida Jim?nez de Oya 0
Juan-Carlos Saiz 0
Miguel A. Mart?n-Acebes
0 , Carlos O?scar S. Sorzano
1 , Javier Ortego
OPEN Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that affects humans and can cause severe neurological complications, including Guillain-Barr? syndrome and microcephaly. Since 2007 there have been three large outbreaks; the last and larger spread in the Americas in 2015. Actually, ZIKV is circulating in the Americas, Southeast Asia, and the Pacific Islands, and represents a potential pandemic threat. Given the rapid ZIKV dissemination and the severe neurological and teratogenic sequelae associated with ZIKV infection, the development of a safe and efficacious vaccine is critical. In this study, we have developed and characterized the immunogenicity and efficacy of a novel ZIKV vaccine based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the ZIKV prM and E structural genes (termed MVA-ZIKV). MVA-ZIKV expressed efficiently the ZIKV structural proteins, assembled in virus-like particles (VLPs) and was genetically stable upon nine passages in cell culture. Immunization of mice with MVA-ZIKV elicited antibodies that were able to neutralize ZIKV and induced potent and polyfunctional ZIKV-specific CD8+ T cell responses that were mainly of an effector memory phenotype. Moreover, a single dose of MVA-ZIKV reduced significantly the viremia in susceptible immunocompromised mice challenged with live ZIKV. These findings support the use of MVA-ZIKV as a potential vaccine against ZIKV.
Zika virus (ZIKV) is a mosquito-borne virus from the family Flaviviridae and the genus Flavivirus1, closely
related with other mosquito-borne viruses with public health importance such as Japanese encephalitis virus
(JEV), West Nile virus (WNV), dengue virus (DENV), and yellow fever virus (YFV). The viral particle has 50nm
in diameter and contains an inner nucleocapsid composed of a linear plus-strand genomic RNA and multiple
copies of the viral capsid (C) protein and an outer host cell-derived lipid bilayer bearing 180 copies each of two
proteins: the viral membrane [M, a cleavage product of the premembrane (prM)] protein and the envelope (E)
protein2,3. The open reading frame encodes a large polyprotein of 3,423 amino acids which is cleaved by viral and
cellular proteases into 10 individual proteins: three structural proteins located at the N-terminal region that form
the infectious virion (C, prM, and E), and seven non-structural proteins located at the C-terminal region involved
in viral replication (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)2,3. ZIKV is transmitted to humans primarily
through the bite of infected mosquitoes from genus Aedes, mainly by A. albopictus and A. aegypti, both widely
distributed throughout the tropical and subtropical regions of the world, with the habitat of A. albopictus extending
further into cool temperate regions2,3. Furthermore, ZIKV can also be transmitted from mother to child during
pregnancy or spread through sexual contact, breastfeeding, or blood transfusion2,3. The multiple modes of ZIKV
transmission make it difficult to develop control strategies against the pathogen.
ZIKV was discovered in Uganda in 1947, but was confined for the first 60 years to an equatorial zone across
Africa and Asia2,3. However, in 2007 a ZIKV outbreak emerged in Yap Island, in the Western Pacific Ocean, and
between 2013 to 2014 a second larger outbreak spread eastward to French Polynesia and other Pacific Islands
that finally reached Latin America in 2015, and disseminated further to North America in 2016; as a
consequence, the World Health Organization (WHO) declared the Public Health Emergency of International Concern
in February 20162,3. Actually, ZIKV is circulating in the Americas, Southeast Asia, and the Pacific Islands, and
represents a potential pandemic threat2,3. In addition, since early 2015, there have been an increasing number
of travel-related imported ZIKV cases in non-endemic countries and it is predicted that a large portion of the
tropical and sub-tropical regions of the globe will have suitable environmental conditions for ZIKV mosquito
transmission. Thus, there is currently a high risk of introducing and establishing new autochthonous transmission
in these areas2,3.
In most cases ZIKV infection causes no symptoms or only a mild self-limiting illness, but recent
epidemiological studies derived from outbreaks in 2007 and 2015 to 2016 linked ZIKV infection to a rising number
of concerning severe neurological diseases, including Guillain-Barr? syndrome and microcephaly2,3. Thus, the
development of a safe and efficacious vaccine against ZIKV is critical given the rapid dissemination of the virus
and the severe neurological and teratogenic sequelae associated with ZIKV infection. There are currently vaccine
candidates in phase I or II clinical trials, and others under development4,5. These vaccine candidates include
various technologies and approaches, such as inactivated ZIKV, recombinant viral vectors, DNA plasmid vaccines,
mRNA-based vaccines, and peptide-based vaccines4,5. Zika vaccine development is mainly based on the whole
inactivated organism or in vectored expression of prM and E structural proteins, as occurred with other flaviviral
vaccines like JEV and DENV.
The highly attenuated poxvirus modified vaccinia virus Ankara (MVA) has been extensively used in numerous
preclinical and clinical trials as a vaccine vector against several infectious diseases, being a cost-effective, safe and
efficacious vector6,7. In addition, recombinant MVA vaccines express high levels of the heterologous antigens, and
are potently immunogenic inducing antigen-specific humoral and T cellular immune responses6,7. Therefore,
MVA should be a potential good vector to develop a vaccine against ZIKV.
Here, we have developed an MVA-based vaccine candidate (termed MVA-ZIKV) expressing the ZIKV prM
and E structural proteins, and have characterized: (i) in vitro, expression of ZIKV proteins in infected cells,
production of virus-like particles (VLPs) and genetic stability of the vector in cell culture at low multiplicity of virus
infection; (ii) in vivo, in immunized mice, ability to activate B- and T-cell responses through the production
of ZIKV neutralizing antibodies in serum, and induction of ZIKV-specific CD8+ T cells in splenocytes; and
(iii) vaccine efficacy after live ZIKV challenge in susceptible immunocompromised mice. The results showed
that MVA-ZIKV produced VLPs, is genetically stable and highly immunogenic in mice, activating B- and T-cell
immune responses with the induction of good titers of neutralizing antibodies against ZIKV, together with potent
and polyfunctional ZIKV-specific CD8+ T cell responses. Interestingly, a single dose of the MVA-ZIKV vaccine
can control ZIKV replication in susceptible immunocompromised mice challenged with ZIKV.
Generation and in vitro characterization of MVA-ZIKV. To generate novel vaccines against ZIKV that
could activate the ZIKV-specific B- and T-cell immune responses, we have generated an MVA-based vaccine
candidate encoding for the ZIKV prM-E structural genes (termed MVA-ZIKV). ZIKV prM-E structural genes of the
ZIKV isolate Z1106033 (Suriname; the most contemporary American isolate available at the time we initiated this
work)8, were inserted into the vaccinia virus (VACV) thymidine kinase (TK) locus of an optimized parental MVA
(termed MVA-?-GFP) containing deletions in the VACV immunomodulatory genes C6L, K7R, and A46R9,10,
and their expression is under the transcriptional control of a novel optimized synthetic Late/Early (pLEO160)
promoter11,12 (Fig.?1a) (see Methods).
The correct generation of MVA-ZIKV was analyzed by PCR using oligonucleotides annealing in the VACV
TK-flanking regions that demonstrated the proper insertion of the ZIKV prM-E structural genes within the
genome of MVA-ZIKV, with no wild-type MVA virus contamination (Fig.?1b). Moreover, the correct nucleotide
sequence of the ZIKV prM-E genes inserted in the VACV TK locus was further confirmed by DNA sequencing
(data not shown).
To demonstrate that MVA-ZIKV constitutively expresses and correctly processes the ZIKV prM-E polyprotein
into prM and E structural proteins, we performed a Western blot analysis of cell extracts from MVA-infective
permissive chicken DF-1 cells, mock infected or infected with MVA-ZIKV, parental MVA-?-GFP, or attenuated
wild-type (WT) MVA (MVA-WT) using specific antibodies that recognize the ZIKV prM and E proteins. The
results proved that MVA-ZIKV correctly expressed the ZIKV prM-E polyprotein that was properly processed
leading to the ZIKV prM and E proteins of expected molecular sizes (Fig.?1c). The presence of two bands in
the western blot using the anti-ZIKV E antibody is compatible with the production of different ZIKV E protein
species that differ in post-translational modification13, being the upper band compatible with the full-length
glycosylated E protein.
To analyze whether expression of ZIKV prM-E structural proteins affects MVA replication in cell culture, we
evaluated the growth kinetics of MVA-ZIKV and MVA-WT in permissive DF-1 cells. Both viruses had a
similar kinetics of viral growth (Fig.?1d), demonstrating that the constitutive expression of ZIKV prM-E structural
proteins does not weaken MVA vector replication under permissive conditions. Moreover, similarly to parental
MVA-WT and as expected, MVA-ZIKV is a viral vector that does not replicate in human HeLa cells (Fig.?1d).
Next, to ensure that MVA-ZIKV is stable and the insert can be maintained in the viral genome without the
loss of the sequence encoding the ZIKV prM-E structural genes, MVA-ZIKV was grown in DF-1 cells infected
at low multiplicity of infection (MOI) for 9 successive passages, and expression of the ZIKV prM and E proteins
was determined by Western blotting (Fig.?1e). The experiment revealed that MVA-ZIKV efficiently expresses the
ZIKV prM and E proteins after consecutive passages. Moreover, analysis by Western blot of the expression of the
ZIKV E protein in 24 individual plaques isolated from MVA-ZIKV at passage 9 showed that 100% of the plaques
correctly expressed the ZIKV E protein (Fig.?1f), demonstrating the high genetic stability of MVA-ZIKV.
Flavivirus prM and E proteins are synthesized at the endoplasmic reticulum (ER)14. Thus, the expression
and intracellular localization of the ZIKV E protein expressed by MVA-ZIKV was studied by confocal
immunofluorescence microscopy in non-permissive HeLa cells infected with MVA-ZIKV and MVA-WT using an
antibody against ZIKV E protein and a specific antibody to detect ER (anti-Calnexin) (Fig.?2a). The results
showed that at 24 h post-infection ZIKV E protein (in green) was highly expressed from MVA-ZIKV-infected
cells and, as expected, co-localized with the ER (Fig.?2a, right panel). Moreover, to determine whether ZIKV E
protein could be detected on the cell surface, HeLa cells were infected with MVA-ZIKV and permeabilized and
non-permeabilized cells were analyzed by confocal immunofluorescence microscopy using an antibody against
ZIKV E protein and the wheat germ agglutinin (WGA) probe to label the surface of fixed cells (Fig.?2b). While the
infected-cell membrane was well observed using the WGA probe, we did not detect labeling of ZIKV E protein.
These results indicate that, as expected, the ZIKV E protein is not present on the surface of infected cells, and
suggest that is release to the medium.
In summary, ZIKV prM and E structural proteins are expressed efficiently by MVA-ZIKV in cultured cells.
MVA-ZIKV produced VLPs. It has been described that coexpression of flaviviral prM and E proteins results
in the production of VLPs [also termed subviral particles (SVPs)]13,15?18. Thus, to test whether MVA-ZIKV could
form VLPs, we investigated their presence in the supernatant of infected cells. Therefore, HeLa cells were infected
with MVA-ZIKV and MVA-WT and at 18 h post-infection, supernatants were concentrated by pelleting through
a 20% sucrose cushion. The analysis by western blot of the concentrated supernatants demonstrated the
presence of ZIKV M and E proteins, confirming their release to the medium (Fig.?3a). Moreover, the detection of
the mature M protein suggests that fully-assembled VLPs could be produced (Fig.?3a). Thus, next we loaded the
concentrated supernatants into a 20?60% w/v sucrose gradient and after ultracentrifugation, fractions were taken
and the amount of protein and sucrose density was analyzed (Fig.?3b). A single peak of protein, determined by
measurement of the absorbance of each fraction at 280 nm, exhibited a density about 1.18 g/cm3 (42% of sucrose)
estimated by refractometry analysis. This value is comparable to that exhibited by other VLPs from different
flaviviruses19?22. The protein peak fraction was then analyzed by negative staining and transmission electron
microscopy to evaluate whether VLPs were observed (Fig.?3c). The results showed that MVA-ZIKV formed smooth
spherical particles of similar size (around 50 nm) and morphology than ZIKV VLPs produced by other ZIKV
vaccines expressing prM-E genes13,15,18,23 (Fig.?3c). Immunogold electron microscopy demonstrated the presence
of ZIKV E protein on the surface of these structures, confirming that these particles were ZIKV VLPs (Fig.?3d).
In summary, MVA-ZIKV expresses ZIKV prM and E proteins that can be assembled into VLPs secreted from
the infected cells.
MVA-ZIKV is highly immunogenic in immunocompetent mice. Neutralizing antibodies against
ZIKV are critical to control ZIKV infection24?26. Thus, to evaluate whether MVA-ZIKV is able to induce
neutralizing antibodies against ZIKV, we determined the plaque reduction neutralization titers (PRNT) against ZIKV
in serum samples obtained at 10 days post-boost from Balb/c mice immunized with MVA-ZIKV or MVA-WT
(negative control group), following a homologous prime/boost immunization protocol (see Methods). The results
showed that individual serum obtained from mice immunized with MVA-ZIKV neutralized ZIKV (PA259459
strain, from Panama) in a dilution-dependent manner (Fig.?4a), compared to serum from MVA-WT-immunized
animals where no neutralization was observed. The elicited mean-value antibody titers induced by MVA-ZIKV
neutralized 90% of ZIKV (PRNT90) at a dilution of 1/50 (Fig.?4b).
Although the specificity of CD8+ T cell responses varies among ZIKV strains, CD8+ T cells might play a
protective role against ZIKV infection27?30. Therefore, to investigate in detail the capability of MVA-ZIKV to
stimulate T cellular immune responses against ZIKV, we next evaluated the ZIKV-specific T cell immune responses
elicited by MVA-ZIKV in Balb/c mice immunized following a homologous prime/boost immunization protocol.
ZIKV E-specific CD4+ and CD8+ T cell immune responses induced by MVA-WT/MVA-WT and MVA-ZIKV/
MVA-ZIKV immunization groups were measured at 10 days post-boost by an intracellular cytokine staining
(ICS) assay, after the stimulation of splenocytes with ZIKV-specific peptide pools spanning the entire ZIKV E
protein (Fig.?5). The results showed that immunization with MVA-ZIKV stimulated robust ZIKV-E-specific
CD8+ T cell immune responses (determined as the percentage of ZIKV E-specific CD8+ T cells producing IFN-?,
TNF-?, and/or IL-2 cytokines, as well as the expression of CD107a on the surface of activated T cells as an
indirect marker of cytotoxicity) (Fig.?5a) (ZIKV-specific CD8+ T cell responses in estimated absolute numbers are
included in Table?S1). MVA-ZIKV elicited total ZIKV-specific immune responses mediated mostly by CD8+ T
cells, with very low levels of ZIKV-specific CD4+ T cells (Fig.?5a). ZIKV-specific CD8+ T cells produced mainly
CD107a, followed by similar levels of IFN-? and TNF-?, and to a minor extent IL-2 (Fig.?5b). The quality of the
ZIKV-specific T cell immune response was defined by the pattern of cytokine production (IFN-?, TNF-?, and/or
IL-2) and its cytotoxic potential (CD107a). Thus, the most representative ZIKV-specific CD8+ T cell populations
induced by MVA-ZIKV were those producing CD107a + IFN-? + TNF-? (triple), CD107a + IFN-? (double) and
CD107a (single), with a high polyfunctional pattern represented by 57% of CD8+ T cells having two, three, or
four functions (Fig.?5c).
Moreover, we also determined the phenotype of the ZIKV-specific CD8+ T cells by evaluating the presence of
CD127 and CD62L surface markers, which define memory subpopulations: T central memory (TCM; CD127+/
CD62L+), T effector memory (TEM; CD127+/CD62L?), and T effector (TE; CD127?/CD62L?) T cells31. The
results showed that immunization with MVA-ZIKV induced ZIKV-specific CD8+ T cells with a phenotype of
TEM (70%) and TE (30%) (Fig.?5d).
In summary, MVA-ZIKV is highly immunogenic in immunized mice inducing neutralizing antibodies against
ZIKV and potent and polyfunctional ZIKV-specific CD8+ T cell immune responses mainly with TEM and TE
MVA-ZIKV controls viral replication in a challenged mouse model. The efficacy of MVA-ZIKV as
a vaccine candidate against ZIKV was studied in mice deficient in the ?/? interferon receptor (IFNAR?/?), a
suitable susceptible mouse model for ZIKV32,33. Thus, six-week-old IFNAR?/? mice were immunized by
intraperitoneal (i.p.) route with MVA-ZIKV (one or two doses) or MVA-WT (one dose; used as a control), at days 0
and/or 14. At day 28 mice were challenged with 104 PFUs of ZIKV (PA259459 strain, from Panama) (Fig.?6a).
Blood was obtained at days 13 and 27 (before challenge) to analyze the neutralizing antibodies in sera, and 2 and
3 days after virus challenge to analyze viremia. The results showed that one or two doses of MVA-ZIKV elicited
good neutralizing antibody titers against ZIKV PA259459 strain, with a second dose significantly increasing the
ZIKV neutralization titer (PRNT90) to around 110 (Fig.?6b). Furthermore, neutralization of another ZIKV strain
from the Asian-American lineage (FSS13025, Cambodia 2010) using serum samples obtained after two doses
of the MVA-ZIKV vaccine showed similar results (PRNT90 149 ? 77), confirming that the elicited neutralizing
antibodies could also neutralize other ZIKV strains. Next, the MVA-ZIKV efficacy was analyzed in immunized
mice after challenge with ZIKV. Intraperitoneal infection of adult IFNAR?/? mice (10-week-old) with the ZIKV
American strain PA259459 did not result in adverse body weight loss or mortality, in spite of induced viremia
(Fig.?6c,d). Along this line, the MVA-ZIKV efficacy, defined as the capacity to control ZIKV replication, was
evaluated at days 2 and 3 post-challenge determining in serum ZIKV viremia by quantitative RT-PCR (Fig.?6c) and
presence of ZIKV infectious virus by plaque assay (Fig.?6d). The results showed that MVA-ZIKV-vaccinated mice
control ZIKV replication as index of significant reduction in levels of ZIKV RNA (Fig.?6c) and of infectious virus
(Fig.?6d), compared to the control group MVA-WT. One or two doses of MVA-ZIKV reduced the ZIKV viremia
and infectious virus by 2.5?4 logs, while MVA-WT-immunized mice developed ZIKV infection, with high levels
of ZIKV RNA and of infectious virus that peaked at day 2 post-challenge (Fig.?6c,d).
In conclusion, MVA-ZIKV is effective in controlling ZIKV replication in a susceptible mouse model for ZIKV
infection, even with a single vaccine dose.
ZIKV is an important emerging flavivirus transmitted by infected mosquitoes from the genus Aedes that can
cause severe complications in humans. The virus has caused recent outbreaks of the disease worldwide and their
future expansion to novel geographical areas is highly possible2,3. Although several ZIKV vaccine candidates have
been generated using different strategies4,5, actually no licensed vaccine exists. These vaccines have been tested in
animals in preclinical trials and in early phase I or II human clinical trials, but although they are immunogenic
many of them required numerous immunizations, have high costs of manufacturing or require the use of
chemical adjuvants. Therefore, novel strategies are necessary to achieve potent and long-lasting protective immune
responses, with less immunizations, better safety profile, and lower costs of vaccine production. An ideal ZIKV
vaccine should (
) be safe, especially for women of child-bearing age; (
) be stable and cost effective to
manufacture, as ZIKV is endemic in developing countries; (
) avoid the addition of chemical adjuvants to minimize
) be effective against all circulating ZIKV strains; and (
) be able to induce a rapid onset of
protective levels of neutralizing anti-ZIKV antibodies as well as T cell responses to control the replication of the virus.
In this report, we have developed a novel, immunogenic and effective ZIKV vaccine candidate (termed
MVA-ZIKV) based on the highly attenuated poxvirus vector MVA expressing the ZIKV prM and E structural
proteins that are assemble into VLPs. MVA-ZIKV induced potent ZIKV-specific B and T cell immune responses
in immunized mice and controlled ZIKV replication in a susceptible immunocompromised mouse model for
ZIKV infection challenged with ZIKV. This is the first report of an MVA-based vaccine against ZIKV
expressing the prM-E structural proteins (the main immunogenic antigens of ZIKV) and forming VLPs. MVA-ZIKV
possesses many of the characteristics needed for an ideal vaccine against ZIKV. MVA-ZIKV is replication
competent in avian cells but replication deficient in human cells, making it safe for humans. The MVA safety profile
has been demonstrated by its use as a variola vaccine during years and by extensive clinical trials testing MVA
recombinants as vaccine vector against different infectious diseases6,7,34, confirming that MVA vaccines are safe
even for immunocompromised individuals, children and elderly. Moreover, a recent study in pregnant macaques
immunized with an MVA vector showed no adverse or teratogenic effects35, opening the possibility of its use
in pregnant women. This observation could be highly relevant in ZIKV vaccination because of severe
sequelae of ZIKV infection during pregnancy. Additionally, it has been reported that VACV induced strong innate
responses during vaccination acting itself as an adjuvant36, eliminating the need of other chemical adjuvants,
minimizing reactogenicity and adverse effects caused by these synthetic substances37,38, and promoting a
better activation of the immune system39. Furthermore, MVA vaccines can be produced easily in primary avian
cells, the most common cells used in the GMP manufactured of MVA lots, and the cost of production and
manufacture is low. Therefore, the use of an MVA vector expressing ZIKV antigens, such as the MVA-ZIKV
reported here, is a promising approach against ZIKV. In addition, the MVA-ZIKV vaccine candidate that we
have developed has a MVA genome more optimized than its parental MVA-WT, due to additional deletions
in 3 MVA immunomodulatory genes (C6L, K7R and A46R), whose deletion enhanced in immunized mice the
HIV-1-specific cellular and humoral immune responses induced by a MVA vector expressing Env, Gag, Pol and
Nef HIV-1 antigens40. Furthermore, one dose of a vaccine against chikungunya virus (CHIKV) based on MVA
with these 3 deletions and expressing CHIKV structural antigens (termed MVA-CHIKV) protected 100% of
animals against CHIKV infection in mice and nonhuman primates9,41. Recently, using this optimized MVA vector
we described MVA-based vaccines that protected against Ebolavirus (EBOV) infection in a susceptible mouse
model10, reinforcing the use of this vaccine platform against emerging viruses, like CHIKV, EBOV and ZIKV.
Our MVA-ZIKV vaccine candidate expresses the ZIKV prM and E structural genes of the ZIKV isolate
Z1106033 (derived from an Asian-American lineage virus, isolated from a patient in Suriname). Although there
are 3 main ZIKV lineages (East African, West African, and Asian-American)42, up to date, only one ZIKV
serotype has been established17. Thus, MVA-ZIKV vaccine should be potentially effective against different ZIKV
strains, due to low heterogeneity between ZIKV lineages. In fact, our results demonstrate that elicited
neutralizing antibodies from MVA-ZIKV are effective against a different ZIKV strain of the Asian-American lineage.
The E protein is the main ZIKV protein involved in receptor binding and fusion, while the M protein is a small
protein that is hidden under the E protein layer43,44. The prM and E proteins are, together with the inactivated
whole viruses, the flaviviral proteins more widely used as vaccine antigens showing an excellent capacity to
protect against this family of virus, including ZIKV4,5. Recent studies comparing in mice and nonhuman primates
the efficacy of diverse vectored-based vaccines against ZIKV, showed that live-attenuated adenoviral vectors
are one of the most potent approaches, even in long-term studies24,45,46. These results confirm the advantage of
live-attenuated recombinant vaccines, a case that can be extrapolated to the MVA-ZIKV vaccine developed here.
Another advantage of the MVA-ZIKV vaccine candidate is that it expresses high levels of ZIKV prM and E
antigens, as observed by Western blot and/or immunofluorescence. This high expression is due to the presence in
the MVA vector of an optimized strong VACV late/early promoter previously described11,12. The ZIKV E protein
expressed by MVA-ZIKV co-localized with the ER (the place where prM-E protein is translocated to dimerize),
supporting their correct subcellular localization. Furthermore, the apparent absence of the ZIKV E protein in the
cell membrane of the MVA-ZIKV-infected cells suggests that the E protein is not exposed in the outer membrane,
but released to the medium. Moreover, we have detected the presence of the ZIKV E protein and the mature M
protein in supernatants of cells infected with MVA-ZIKV, confirming their release to the medium. Remarkably,
the analysis by immunogold electron microscopy of the purified supernatants demonstrated that MVA-ZIKV
produced VLPs, similarly to other ZIKV vaccines or flavivirus expressing the prM-E genes13,15,18,23. Furthermore,
an additional advantage as a vector is that MVA-ZIKV is highly stable in cell culture, maintaining the expression
of ZIKV antigens at least during 9 continuous passages at low MOI, with 100% of the plaques picked at passage 9
expressing the correct size ZIKV antigens.
The adaptive immune response against ZIKV plays an essential role in regulating ZIKV infection and
preventing the spread of the virus to key organs, like the brain and testes27,46, especially when type I IFN response
is reduced, as in human ZIKV infection47. It has been described that neutralizing antibody titers higher than 10
correlated with protection after vaccination with most of the licensed flavivirus vaccines48, and adoptive transfer
studies suggest that vaccine-elicited antibodies, even at low titers, are sufficient for protection against ZIKV
challenge in mice and nonhuman primates24?26. Our MVA-ZIKV, after 2 doses and at 10?13 days post-boost, induced
antibody titers (PRNT90) of 50 or higher than 100 in WT or IFNAR?/? mice, respectively, in the range of a good
and immunogenic vaccine against ZIKV.
On the other hand, to date relatively little is known regarding the T cell response to ZIKV infection. It has been
recently shown that adoptive transfer of ZIKV-specific CD8+ T cells prevented disease in immunocompromised
susceptible mice27,29. Moreover, given the neurotropism of ZIKV and its presence in fetal brain tissues and
cerebrospinal fluid in both humans and animal models49, CD8+ T cells may have a key role in clearing ZIKV from
the central nervous system (CNS) and, thus, in preventing or mitigating neurological complications. A recent
study in pregnant macaques infected with ZIKV and then treated with a cocktail of ZIKV-neutralizing human
monoclonal antibodies (mAbs) at the peak of viremia, showed that while the mAbs can be effective in clearing
the virus from the maternal sera of treated nonhuman primates, it is not sufficient to fully stop vertical
transmission50. This finding suggest the importance of CD8+ T cell immune response against ZIKV infection, especially
when the immune system integrity is compromised, as during pregnancy51. These results suggest that a vaccine
eliciting ZIKV-specific CD8+ T cell responses could contribute effectively at preventing disease and clearing the
virus from the CNS. Up to now only few vaccine candidates have described the induction of ZIKV-specific T cell
responses towards E protein; reporting mainly a modest activation24,25,46,52. In our case and using
immunocompetent mice, MVA-ZIKV is able to induce potent and polyfunctional ZIKV-specific CD8+ T cell immune responses,
as natural ZIKV infection does29, indicating that MVA-ZIKV could be a promising vaccine candidate against
ZIKV. Furthermore, the low CD4+ T cell response that we observed was consistent with a report where
appearance of CD4+ T cell responses in ZIKV-infected rhesus monkeys was not found until production of antibodies
and CD8+ T cell responses, at 2 weeks post-infection53.
Therefore, all these results highlight the need for the development of vaccines that drive a potent T cell and
humoral antibody responses, as MVA-ZIKV, in order to protect against ZIKV infection and spread.
Current immunocompetent mouse models for ZIKV are not susceptible to viremia or lethal outcome. Hence,
to analyze the efficacy of MVA-ZIKV we used a type I IFN receptor-knockout mouse model (IFNAR?/?) that is
extensively used as a challenge model for ZIKV vaccine candidates32,33. As it happened in immunocompetent
Balb/c mice, IFNAR?/? mice immunized with one or two doses of MVA-ZIKV produced ZIKV-neutralizing
antibodies (against ZIKV PA259459 strain or against other isolates such as FSS13025) and controlled ZIKV viral
replication after a challenge with live ZIKV PA259459 strain Panama, representative of the circulating virus
during the American epidemic. The potent generation of neutralizing antibodies by MVA-ZIKV could be due to the
production of VLPs, which are efficiently recognized by the B cells54, leading to an MHC-II upregulation that will
favor the production of high levels of neutralizing antibodies against ZIKV. VLPs are highly immunogenic and
therefore are very important to improve the ZIKV-specific immune responses in vivo, as it has been reported with
VLPs from ZIKV or other viruses that induced high titers of neutralizing antibodies which correlate with
protection13,15,18,23,55. From a vaccine point of view, we suggest that in order to further enhance in vivo the production of
ZIKV neutralizing antibodies, it might be convenient to perform immunizations with the combined MVA-ZIKV
and purified VLPs as immunogens.
We observed that after ZIKV challenge infection immunized IFNAR?/? mice (10-week-old at the moment of
challenge) did not loss body weight or have mortality during 15 days post-challenge. Our results are consistent
with data obtained in adult IFNAR?/? mice (10?11-week-old) challenged with other ZIKV strains belonging to
the same genetic lineage than PA259459 strain that did not develop disease and had higher survival rates than
young mice challenged with the same ZIKV strain32,33,56,57. Accordingly, we selected this non-lethal challenge
model for the evaluation of vaccine efficacy because it resembles better the ZIKV infection in humans, which
usually provokes a mild disease that is not fatal.
The control of ZIKV infection obtained after administration of one or two doses of MVA-ZIKV was in the
range between 2.5 and 4 log virus reduction, highlighting the efficacy of the vaccine. We do not know the
contribution of B and T cell responses in the control of ZIKV infection in this mouse model. However, considering the
potent ZIKV-specific CD8+ T cellular immunogenicity and humoral immune responses induced by MVA-ZIKV
in the immunocompetent mouse model, we suggest that in a similar way neutralizing antibodies and CD8+ T
cells should both contribute to the effective reduction of viral load observed in IFNAR?/? mice and this vaccine
would be an effective approach to protect against ZIKV. Further studies will be needed to define in this system
the independent role of B and T cell responses elicited by MVA-ZIKV in virus protection. Remarkably, the fact
that MVA-ZIKV induced antibodies that can neutralize other ZIKV strain (FSS13025, Cambodia 2010), the high
homology among ZIKV structural proteins from different isolates and the existence of only one ZIKV serotype17
support that the protection we observed could be likely extended to other different ZIKV strains. The protection
of mice vaccinated with MVA-ZIKV against other distant ZIKV strains will be tested in future studies.
In summary, we have developed a novel and promising ZIKV vaccine candidate (MVA-ZIKV) that produced
VLPs and in mice induced ZIKV-specific neutralizing antibodies and potent CD8+ T cell immune responses,
being strongly effective in reducing ZIKV viremia after a challenge with ZIKV.
Ethics statement. The immunogenicity animal studies were approved by the Ethical Committee of Animal
Experimentation (CEEA) of Centro Nacional de Biotecnolog?a (CNB, Madrid, Spain) and by the Division of
Animal Protection of the Comunidad de Madrid (PROEX 331/14) and were conducted at the CNB. The
efficacy animal studies were approved by the CEEA of Instituto Nacional de Investigaci?n y Tecnolog?a Agraria
y Alimentaria (INIA, Madrid, Spain) and by the Division of Animal Protection of the Comunidad de Madrid
(PROEX 187/17) and were conducted in the biosafety level 3 laboratory at Centro de Investigaci?n en Sanidad
Animal (CISA-INIA, Madrid, Spain). Animal procedures were conformed to international guidelines and to the
Spanish law under the Royal Decree (RD 53/2013). Animals were maintained and handled according to the
recommendations of the CNB-CSIC and CISA-INIA institutional Ethics Committees.
Cells. HeLa cells (a human epithelial cervix adenocarcinoma; ATCCCCL-2), DF-1 cells (a spontaneously
immortalized chicken embryo fibroblast (CEF) cell line; ATCCCRL-12203) and primary CEF cells (obtained
from specific-pathogen-free 11-day-old eggs; MSD, Salamanca, Spain) were grown in Dulbecco?s modified Eagle?s
medium (DMEM) and 10% heat-inactivated fetal calf serum (FCS) (Gibco-Life Technologies), as previously
described58. Vero cells (a kidney epithelial cell line from African green monkey; ATCCCCL-81) were grown in
Eagle?s minimal essential medium (EMEM) and 5% heat-inactivated fetal bovine serum (Linus), as previously
described59. Cell cultures were kept at 37 ?C and 5% CO2 in a humidified incubator.
Viruses. The parental VACV used for the generation of the recombinant MVA-ZIKV is an attenuated
MVA-WT that was modified by inserting the green fluorescent protein (GFP) gene into the VACV TK locus and
by deleting the immunomodulatory VACV genes C6L, K7R, and A46R (parental virus termed MVA-?-GFP)9,10.
Then, the GFP gene present in the TK locus of MVA-?-GFP was replaced by the ZIKV prM-E genes to generate
the MVA-ZIKV vaccine candidate (see later). We also include as control the MVA-WT. All MVAs were grown
in primary CEF cells, purified, and titrated at least two times, as previously described60. All MVAs were free of
contamination with mycoplasma, bacteria or fungi. ZIKV PA259459 and FSS13025 strains were isolated from an
infected human in Panama in 2015 and in Cambodia in 2010, respectively. Both ZIKV isolates were propagated
and titrated in semisolid agarose medium using Vero cells, as previously described61.
ZIKV antigens. The ZIKV antigens inserted in the MVA-ZIKV vaccine candidate are the prM and E
structural genes of ZIKV isolate Z1106033 (derived from an Asian-American lineage virus, isolated from a patient
in Suriname at the onset of the late-2015 expansion of the virus in the Americas; GenBank accession number:
KU312312)8. The prM and E are preceded by the last 18 amino acids of the C-terminal hydrophobic stretch
of C-protein that acts as a signal peptide for the proper translocation of the prM protein into the lumen of the
ER. The ZIKV prM-E sequence was chemically synthesized and codon optimized for human cell expression by
GeneArt? (Thermo Fisher Scientific).
Construction of pLEOLZ-ZIKV plasmid. The synthesized ZIKV prM-E genes were subcloned into the
plasmid transfer vector pLEOLZ (Centro Nacional de Biotecnolog?a) using the Gibson Assembly? technology
(New England Biolabs), to obtain the pLEOLZ-ZIKV plasmid. The plasmid was amplified in ElectroMAX?
DH10B? Cells (Thermo Fisher Scientific) and purified using a QIAGEN Plasmid Maxi Kit (Qiagen). Its correct
generation was confirmed by DNA sequencing. Plasmid transfer vector pLEOLZ-ZIKV was used for the
generation of recombinant MVA-ZIKV and allows the insertion of the ZIKV prM-E antigens in the TK locus of parental
MVA-?-GFP by homologous recombination. It contains a novel synthetic VACV promoter termed pLEO16011,12,
a multiple-cloning site, the human codon-optimized prM-E ZIKV genes introduced between the VACV TK-L
and TK-R flanking regions, and the selectable marker genes for ampicillin and ?-galactosidase (LacZ gene). The
LacZ gene is inserted among two repetitions of the left TK-flanking region, allowing the deletion of the reporter
LacZ gene from the final recombinant virus by homologous recombination after consecutive plaque purification
Generation of recombinant MVA-ZIKV. MVA-ZIKV was generated using MVA-?-GFP as parental virus
and pLEOLZ-ZIKV as a plasmid transfer vector, using an infection/transfection protocol9,10. The recombinant
virus obtained (termed MVA-ZIKV) was then grown in CEF cells, purified and titrated by plaque
PCR analysis of MVA-ZIKV. The correct generation and purity of recombinant MVA-ZIKV was confirmed
by PCR using oligonucleotides TK-L and TK-R, annealing in the VACV left and right TK-flanking regions,
respectively, and allowing the amplification of the ZIKV insert, as previously described58,62,63. Furthermore, the
correct ZIKV insert was also confirmed by DNA sequencing.
Analysis of MVA-ZIKV virus growth. To study the virus growth profile of MVA-ZIKV in comparison to
that of MVA-WT, monolayers of permissive DF-1 cells or non-permissive HeLa cells were infected at 0.01 PFU/
cell with MVA-WT or MVA-ZIKV, as previously described9,10. At different hours post-infection (hpi) (0, 24, 48,
and 72) cells were harvested and virus titers in cell lysates were determined by a plaque immunostaining assay in
DF-1 cells, as previously described60.
Expression of ZIKV proteins from MVA-ZIKV by Western blot. To test the expression of the ZIKV
antigens present in MVA-ZIKV, monolayers of DF-1 cells were mock infected or infected at 5 PFU/cell with
MVA-WT, MVA-?-GFP, or MVA-ZIKV. At 24 hpi cells extracts were loaded and separated in 10% SDS-PAGE,
and then analyzed by Western blotting with a rabbit polyclonal antibody against ZIKV prM (Genentex; diluted
1:2,000) or a mouse monoclonal antibody against ZIKV E (BioFront Tech; diluted 1:5,000) to detect the ZIKV
prM and E proteins. As loading controls, a rabbit anti-?-actin antibody (Cell Signaling; diluted 1:1,000), and
a rabbit anti-VACV E3 antibody (Centro Nacional de Biotecnolog?a; diluted 1:1,000) were used. Anti-mouse
or anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies (Sigma; diluted 1:2,000 or 1:5,000,
respectively) were used as secondary antibodies. The immune complexes were detected with an HRP-luminol
enhanced-chemiluminescence system (ECL Plus, GE Healthcare).
Genetic stability of MVA-ZIKV. The genetic stability of recombinant MVA-ZIKV was examined as
previously described9,58,63. MVA-ZIKV (P2 stock) was continuously grown at low MOI in DF-1 cells during 9 passages
and then 24 individual plaques were picked from virus derived from passage 9. Next, virus from the 9 passages
and the 24 individual plaques from passage 9 were used to infect cells and the expression of ZIKV prM and E
proteins was checked by Western Blot as described above.
Expression of ZIKV E protein from MVA-ZIKV by confocal immunofluorescence microscopy.
Immunofluorescence experiments were performed in HeLa cells at 24 hpi, as previously described9,63. We used
as ER marker a rabbit polyclonal anti-calnexin antibody (BioNova; diluted 1:200) and a mouse monoclonal
antibody against ZIKV E protein (BioFront Tech; diluted 1:200) to detect ZIKV E protein in infected
permeabilized cells. Anti-ZIKV E and anti-calnexin were detected with mouse secondary antibodies conjugated with the
fluorochrome Alexa Fluor 488 (green) and Alexa Fluor 594 (red), respectively (Invitrogen; diluted 1:500). We
used DAPI (4?,6?-diamidino-2-phenylindole; Sigma) to stain the cell nuclei. Furthermore, permeabilized and
non-permeabilized infected HeLa cells were stained with WGA probe conjugated to the red fluorescent dye Alexa
Fluor 594 (Invitrogen; diluted 1:200) and the mouse monoclonal antibody against ZIKV E protein (BioFront
Tech; diluted 1:200) to label the cell membrane and the ZIKV E protein, respectively. Images of sections of the
cells were taken with a Leica TCS SP5 microscope.
Detection of VLPs. The detection of VLPs in the supernatants of cells infected with MVA-ZIKV was done as
previously described10,64. Briefly, HeLa cells were infected with MVA-ZIKV or MVA-WT at an MOI of 10 PFU/
cell. Supernatants were collected at 18 hpi, and were concentrated and purified by ultracentrifugation through a
20% sucrose cushion at 25,000 rpm in a Beckmann SW28 rotor for 2 h. Then, the pellet was resuspended in 1ml
of PBS1X, and an aliquot of 50 ?l was collected to analyse by western blot the presence of ZIKV M and E proteins.
Next, the rest of the sample was loaded into a 20?60% w/v sucrose gradient and ultracentrifugated at 35,000 rpm
in a Beckmann SW41 rotor for 18 h. Fractions of 500 ?l were taken and the amount of protein and sucrose
density was analyzed. The fraction with higher amount of protein (fraction 14) was then used for dialysis through
a 0.025-? m-pore-size membrane filter (Merck Millipore) for 12 h. Then, 20? l of the sample was adsorbed to
carbon-coated collodion films mounted on 400-mesh/inch nickel grids (Aname, Spain), and were processed as
previously described10,64. Samples were incubated with a mouse anti-ZIKV E primary antibody (BioFront Tech;
diluted 1:20), an anti-IgG secondary antibody coupled to 10-nm colloidal gold beads (BBInternational; diluted
1:40), and then stained with 2% uranyl acetate (Aname, Spain). Then, pictures were taken using a transmission
electron microscope (JEOL JEM-1011; Centro Nacional de Biotecnolog?a, Spain) equipped with a ES1000W
Erlangshen charge-coupled-device (CCD) camera (Gatan Inc.) at an acceleration voltage of 40 to 100 kV.
Mouse immunogenicity study. Female Balb/cOlaHsd mice (6-8-week-old) were purchased from
ENVIGO Laboratories and stored in the animal facility of the CNB (Madrid, Spain). A homologous MVA prime/
MVA boost immunization protocol was done to evaluate the immunogenicity of the MVA-ZIKV vaccine
candidate. Groups of animals (n = 3) were immunized with 1 ? 107 PFUs of MVA-WT or MVA-ZIKV by the i.p. route
in 200 ?l of PBS. Two weeks later, animals received a second dose of 2 ? 107 PFUs of MVA-WT or MVA-ZIKV.
Ten days post-boost, mice were sacrificed using CO2. Blood from of each individual mouse was collected and
processed to obtain sera samples to analyze the titers of neutralizing antibodies against ZIKV; and spleens were
extracted and processed to measure T cell immune responses to the ZIKV E antigen by an intracellular cytokine
staining (ICS) assay. No adverse effects were detected in mice immunized with MVA-ZIKV or MVA-WT.
Peptides. A ZIKV E peptide pool of the ZIKV PRVABC59 strain (GenPept: AMZ03556) was used in the
immunogenicity analysis. Each purified peptide of the ZIKV E peptide pool is at 1 mg per vial, and was obtained
through BEI Resources (National Institute of Allergy and Infectious Disease, National Institutes of Health, USA).
They spanned the entire ZIKV E protein as consecutive 15-mers overlapping by 12 amino acids.
ICS assay. The magnitude, polyfunctionality, and phenotype of the ZIKV-specific adaptive T cell responses
were analyzed by ICS, as previously described9,40,65,66. After spleen processing, splenocytes (depleted of red blood
cells) were stimulated with 5 ?g/ml of the ZIKV E peptide pool. Then, cells were stained for the surface markers,
fixed, permeabilized (Cytofix/Cytoperm kit; BD Biosciences), and stained intracellularly with the appropriate
fluorochrome-conjugated antibodies, as previously described9,40,65,66.
Plaque reduction neutralization assay (PRNT). Titers of neutralizing antibodies against ZIKV present
in the sera of immunize mice were determined by a PRNT assay using Vero cells, as previously described64. Titers
of neutralizing antibodies were expressed as the reciprocal of the serum dilution that inhibited plaque formation
by 90% (PRNT90), relative to samples incubated with negative control sera.
IFNAR?/? challenge study. To evaluate the efficacy of MVA-ZIKV, a ZIKV challenge study was performed
in a biosafety level 3 laboratory at the CISA-INIA, Madrid, Spain. Groups of female IFNAR?/? mice (n = 10 mice/
group; 6-week-old) were immunized with 2 ? 107 PFUs of MVA-WT (one dose, at day 0) or MVA-ZIKV (one or
two doses, at days 0 and 14, respectively) by the i.p. route. At days 13 and 27 post-prime immunization (13 days
after one or two immunizations, respectively) mice were bled from check bleedings and serum samples were used
to determine the levels of neutralizing antibodies against ZIKV by a PRNT assay, as described above. No adverse
effects were observed in mice immunized with MVA-ZIKV or MVA-WT. Four weeks after the first immunization,
mice (at an age of 10 weeks) were challenged with 104 PFUs of ZIKV (PA259459, strain Panama) via the i.p. route.
After challenge, blood samples were collected at days 2 (n = 5) or 3 (n = 5), and ZIKV viremia was determined by
quantitative RT-PCR and by a plaque assay as described below. Mice were checked daily after challenge for signs
of disease and body weight during 15 days. At 15 days post-challenge mice were sacrificed.
Analysis of ZIKV viremia. Viral RNA was automatically extracted from serum samples using QIAmp?
Viral RNA Mini Kit (Qiagen) and a QIAcube apparatus (Qiagen). The amount of viral RNA was
determined by real-time fluorogenic RT-PCR in a Rotorgene 3000 equipment (Corbett Research) using the High
Scriptools-Quantimix Easy probes kit (Biotools) and a previously described primer set and probe specific for
ZIKV67. Genomic equivalents to PFU/ml were calculated by comparison with 10-fold serial dilutions of ZIKV
RNA extracted from previously titrated samples. The amount of ZIKV infectious particles in serum samples
from infected mice was also determined by titration of 10-fold dilutions of serum samples in Vero cells grown in
semisolid agarose medium.
Data analysis and statistical procedures. Statistical analysis of the ICS data was realized as previously
described62,68, using an approach that corrects measurements for the medium response (RPMI), with calculation
of confidence intervals and P values. Only antigen response values significantly larger than the corresponding
RPMI values are represented. Background values were subtracted from all values used to allow analysis of
proportionate representation of responses. The statistical significance of neutralization measurement (PRNT90) in Balb/c
sera was determined by an unpaired t-test, and in IFNAR?/? mice by analysis of the variance (ANOVA) applying
Bonferroni?s correction for multiple comparisons. The statistical significance of viremia was also determined by
ANOVA applying Bonferroni?s correction for multiple comparisons. Statistically significant differences in the
figures are denoted by *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001).
This research was supported by Spanish grants SAF2013-45232-R and SAF2017-88089-R (AEI/MINECO/
FEDER-EU) to Mariano Esteban; AGL2014-56518-JIN (MINECO) to Miguel A. Mart?n-Acebes; and
RTA2001300013-C04-01-E (MINECO) and ZIKA-BIO-2016-02 (INIA) to Juan-Carlos Saiz. Patricia P?rez received a
Formaci?n de Personal Investigador PhD fellowship from the Spanish Ministry of Health and Education. We
thank Cristina S?nchez Corzo and Victoria Jim?nez for expert technical assistance in cell culture, virus growth
and purification. We also thank Silvia Guti?rrez and Ana O?a from the CNB Advanced Light Microscopy
Facility for their valuable support in confocal microscopy experiments, Cristina Pati?o from the CNB Electron
Microscopy Service for their valuable work with the electron microscopy and Iv?n Jare?o from the CNB Animal
Facilities for their help and assistance.
P.P. constructed and characterized the vaccine in vitro and performed the immunogenicity study included in
Fig. 5. M.Q.M., A.L.-F. and J.G.-A. help in the generation, in vitro characterization and immunogenicity of the
vaccine. N. JdO., A.-B.B., E.E.-R. and M.A.M.-A. analyzed the neutralizing antibodies included in Figs 4 and 6
and performed the efficacy trial showed in Fig. 6. C.O.S.S. analyzed data in Fig. 5. J.O. provided the IFNAR?/?
animals. J.-C.S. contributed to the study design. P.P., M.E., M.A.M.-A. and J.G.-A. conceived and designed the
experiments and wrote the manuscript. M.E., M.A.M.-A. and J.G.-A. supervised the work. All authors approved
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-35724-6.
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