The vaccinia virus based Sementis Copenhagen Vector vaccine against Zika and chikungunya is immunogenic in non-human primates

The Sementis Copenhagen Vector (SCV) is a new vaccinia virus-derived, multiplication-defective, vaccine technology assessed herein in non-human primates. Indian rhesus macaques (Macaca mulatta) were vaccinated with a multi-pathogen recombinant SCV vaccine encoding the structural polyproteins of both Zika virus (ZIKV) and chikungunya virus (CHIKV). After one vaccination, neutralising antibody responses to ZIKV and four strains of CHIKV, representative of distinct viral genotypes, were generated. A second vaccination resulted in significant boosting of neutralising antibody responses to ZIKV and CHIKV. Following challenge with ZIKV, SCV-ZIKA/CHIK-vaccinated animals showed significant reductions in viremias compared with animals that had received a control SCV vaccine. Two SCV vaccinations also generated neutralising and IgG ELISA antibody responses to vaccinia virus. These results demonstrate effective induction of immunity in non-human primates by a recombinant SCV vaccine and illustrates the utility of SCV as a multi-disease vaccine platform capable of delivering multiple large immunogens.


INTRODUCTION
Poxvirus-based vaccine vector systems have a number of attractive features including (i) the ability to accommodate large recombinant immunogen payloads (at least 25,000 base pairs), (ii) a capacity for cold chain-independent distribution, (iii) the lack of DNA integration and (iv) the potential for needle-free vaccine delivery (reviewed in ref. 1 ). A range of vaccine vector systems based on vaccinia virus (VACV) and other poxviruses have been developed, with several sold as products and many more in human clinical trials 1,2 . These include NYVAC 3 , ALVAC 4 , fowlpox 5 and Modified Vaccinia Ankara (MVA) 6 , with a large series of recombinant MVA (rMVA) vaccines evaluated in non-human primate (NHP) studies 2,7 and in human clinical trials 1,8 . In humans and NHPs, rMVA vaccines are generally effective at boosting immune responses, but are often poor as stand-alone vaccines for induction of effective immune responses to recombinant vaccine antigens in naive individuals. Heterologous prime-boost strategies (e.g., DNA prime and rMVA boost) have been widely adopted to overcome this limitation 1,7,9,10 . MVA was rendered replication defective in mammalian cells by 572 passages of VACV in primary chicken embryo fibroblasts (CEFs) and, towards the end of the global smallpox, vaccination campaign was used in ≈120,000 people with no significant side effects. MVA is currently sold as a smallpox vaccine as IMVANEX/IMVAMUNE (by Bavarian Nordic) 8,11 . Manufacture of rMVA/MVA vaccines has relied on the use of CEFs, although EB66, a duck embryo-derived cell line, may be used in the future 12 . The other aforementioned poxvirus vectors similarly use CEFs for production.
The Sementis Copenhagen Vector (SCV) represents a new vaccine vector technology based on the Copenhagen strain of VACV. SCV is unable to generate infectious viral progeny in vaccine recipients due to a targeted deletion of D13L, a gene encoding the essential viral assembly protein, D13. Recombinant SCV vaccines are produced in Chinese Hamster Ovary (CHO) cells modified to express D13 and the host range protein CP77. CHO cells are widely used in the manufacture of biologics and provide a significant advantage over CEFs that have traditionally been used for the manufacture of VACV-based vaccines 1,13,14 . A singleconstruct, multi-pathogen SCV vaccine encoding the structural gene cassettes of chikungunya virus (CHIKV) and Zika virus (ZIKV) (SCV-ZIKA/CHIK) was recently shown to be immunogenic and protective against both viruses in a series of mouse challenge models after a single vaccination 14 . An SCV vaccine was also shown to protect against ectromelia virus (a mouse model of smallpox) and to be non-pathogenic in SCID mice (a mouse model of lethal progressive VACV infection) 13 .
The largest outbreak of CHIKV ever recorded started in 2004 and reached >100 countries in four continents, with >10 million cases of primarily rheumatic disease, with mortality rate estimates ranging from 0.024 to 0.7% of CHIKV cases 15 . In 2016, the World Health Organization declared the ZIKV pandemic a public health emergency of international concern. ZIKV is the aetiological agent of Congenital Zika Syndrome (CZS), a spectrum of primarily neurological abnormalities (including microcephaly) in newborns arising from ZIKV infection of pregnant mothers, with >4300 CZS cases reported in Brazil in 2016 16 . A considerable international 1 effort has been underway to develop vaccines against both CHIKV 17 and ZIKV 18 . A combination ZIKV and CHIKV vaccine is deemed attractive as these two arboviruses (i) co-circulate in many parts of the world 19 , (ii) can be transmitted by the same mosquito vectors and (iii) can co-infect both humans 20 and mosquitoes 21 . Single-construct multi-pathogen vaccines such as the SCV-ZIKA/CHIK vaccine 14 (and the recently described vesicular  stomatitis virus ZIKV/CHIKV vaccine 22 ) provide simplified manufacturing and formulation 23 , with multi-pathogen vaccines generally providing reduced 'shot burden', increased compliance and reduced costs 1,24 .
Herein we describe the evaluation of a SCV vaccine in NHPs (Indian rhesus macaques, Macaca mulatta) and show that SCV-ZIKA/CHIK induces antibody responses to CHIKV, ZIKA and VACV, and provides protection against ZIKV challenge.

SCV vaccination
NHPs were vaccinated once with 10 7 pfu (n = 5) or twice with 10 8 pfu of SCV-ZIKA/CHIK (n = 5), or twice with 10 8 pfu of the SCVcontrol vaccine (n = 4) (Fig. 1a). Animals were vaccinated via an intramuscular injection into the right quadriceps (single site) in a volume of 0.5 ml. No fever or loss of body weight was observed post-vaccination (Supplementary Table 1). No adverse events were noted, except for NHP 5548, who developed a rash by day 35 in the inguinal area, extending from the lower abdomen to the upper thighs. The rash failed to resolve throughout the study and was bilateral, moderately red, flat and similar to those sometimes seen in naive NHPs. When NHPs sit on perches with their legs drawn into their bodies for long periods, moisture and reduced airflow to these areas are likely contributors. This event was thus unlikely to have been related to vaccination, although a rash is an uncommon (≥1/1000 to <1/100) adverse reaction after vaccination of humans with IMVANEX (MVA) 25 .
A positive control vaccine comprising formalin-inactivated PRVABC59 given twice intramuscularly (i.m.) to two NHPs generated high levels of neutralising antibody responses to ZIKV PRVABC59 ( Supplementary Fig. 1) as described previously 26 .
ZIKV challenge All NHPs were challenged with ZIKV PRVABC59 and the ZIKV RNA copy numbers in serum (indicative of viraemias) were determined by quantitative reverse-transcriptase PCR (qRT-PCR) (Fig. 1a). Serum ZIKV RNA copy numbers in both SCV-ZIKA/CHIK-vaccinated groups (Fig. 1b, c) were lower than in the SCV-control vaccine group (Fig. 1d), with both reaching significance using area under the curve (AUC) calculations (Fig. 2b). Two NHPs in the SCV-ZIKA/ CHIK 10 7 pfu vaccine group showed serum ZIKV RNA copy numbers above the lower limit of reliable quantification (LLoRQ) determined to be 860 ZIKV RNA copies/ml, indicating that these animals had low level viremias. These animals were NHP 5552 that exceeded the LLoRQ for 3 days and NHP 5550 that exceeded the LLoRQ for 1 day (Supplementary Fig. 2). These two NHPs also had the lowest neutralising titres of the SCV-ZIKA/CHIK-vaccinated animals on the day of challenge (Fig. 1b, Day 70). None of the NHPs vaccinated with 2 × 10 8 pfu of SCV-ZIKA/CHIK showed serum ZIKV RNA copy numbers above the LLoRQ, whereas all NHPs receiving the SCV-control vaccine showed serum ZIKV RNA copy numbers above the LLoRQ (Supplementary Fig. 2). As expected 26 , the two NHPs vaccinated with formalin-inactivated ZIKV PRVABC59 vaccine showed no serum ZIKV RNA copy numbers above the LLoRQ (Supplementary Figs. 1 and 2).
Notwithstanding the uncertainties of qRT-PCR determinations below the LLoRQ, for all SCV-ZIKA/CHIK-vaccinated NHPs (n = 10), the AUC for ZIKV RNA copies/ml ( Supplementary Fig. 2) inversely correlated (p < 0.001) with neutralisation titres (Fig. 2c). This analysis supports the view that neutralising antibodies play a protective role against ZIKV infection 27 , although it does not preclude a protective role for T cells 28,29 .
CHIKV antibody responses Three CHIKV genotypes are recognised; the West African (WA); the East, Central and South African (ECSA), and the Asian. An Indian Ocean sub-lineage (IOL) arose from the ECSA lineage during the Indian Ocean outbreak. Asian genotype viruses (introduced into the Caribbean in 2013) were responsible for most of the disease in the Americas, with the American viruses forming a sub-lineage within the Asian genotype 30 13,14 . A dendogram of the E2 protein sequences of these isolates is shown in Supplementary Fig 3. E2 is a major target of neutralising antibodies [32][33][34] SCV-ZIKA/CHIK vaccination resulted in the generation of neutralising antibodies to all four CHIKV isolates ( Fig. 3a-b), whereas responses following SCV-control vaccination were minimal (Fig. 3c). The individual titration curves are shown in Supplementary Fig 4. On day 35 after one vaccination with 10 7 pfu of SCV-ZIKA/CHIK, the responses to the two Asian isolates (R99659 and AF15561) were 5.6-fold lower than those against the IOL and WA isolates (

VACV antibody responses
We have previously shown in mice the induction of anti-VACV antibody responses by SCV vaccination and protection against ectromelia virus (mouse pox) challenge 13 , suggesting the potential for SCV to also be used as a smallpox vaccine. The VACV neutralising (Fig. 4a, c, e) and IgG enzyme-linked immunosorbent assay (ELISA) antibody ( Fig. 4b, d, f) titres induced by SCV vaccination were thus determined. A single vaccination with 10 7 pfu only produced detectable neutralising antibody responses in 1 NHP (NHP557), with low IgG ELISA response seen on days 20 and/ or 40, but below detection by day 70 post vaccination ( Fig. 4a-b). After 2 i.m. vaccinations with 10 8 pfu of SCV vaccines (given week 0 and 5), the VACV neutralisation titre reached a mean reciprocal 50% Plaque Reduction Neutralisation test (PRNT 50 ) titre of 79 ± SEM 24 (n = 9) by day 70, taking both the 2 × 10 8 SCV-ZIKA/CHIK (Fig. 4c) and SCV-control groups (Fig. 4e) together. The VACV ELISA IgG titres largely paralleled the neutralising antibody responses, with both the 2 × 10 8 SCV-ZIKA/CHIK (Fig. 4d) and the SCV-control groups (Fig. 4f) reaching similar mean titres by day 70 with an overall (both groups) mean reciprocal endpoint ELISA titre of 800 ± SEM 163 (n = 9).

DISCUSSION
We present herein a NHP immunogenicity study for an SCV vaccine. SCV-ZIKA/CHIK vaccination induced anti-CHIKV, anti-ZIKV and anti-VACV antibody responses and mediated protection against ZIKV challenge.
We show herein that SCV-ZIKA/CHIK vaccination was able to reduce post-challenge serum ZIKV RNA copy numbers to levels below those that can reliably be detected, except in two NHPs that received one dose of 10 7 pfu SCV-ZIKA/CHIK (NHPs 5552 and 5550). Sterilising immunity (nominally defined as no viral RNA detected) was not achieved for any NHP, even those vaccinated with the positive control vaccine 26 (Supplementary Fig. 2). Whether sterilising immunity is required to prevent transplacental fetal brain infection in pregnant women remains unclear. Whether low levels of placental or fetal infection could give rise to neonatal disabilities, or disabilities that might manifest later in life, remains similarly unclear 35 . The highly sensitive nature of qRT-PCR would make such sterilising immunity difficult to achieve, with very high levels of neutralising antibodies likely required 36 . Attaining and sustaining such high levels in women of child-bearing age would represent a considerable challenge, especially given the additional safety concerns in this population. A consensus view will have to await vaccine studies in pregnant NHPs, with the a hope being that a vaccine that is able to reduce substantially ZIKV replication will largely prevent or significantly ameliorate the rate and severity of CZS. Such a hope is supported by the success of vaccination campaigns against rubella 37 (family Matonaviridae, genus Rubivirus) and bovine viral diarrhoea virus (family Flaviviridae, genus Pestivirus) 38 .
A concern for ZIKV vaccines is that they may induce antibodydependent enhancement (ADE) of dengue virus (DENV) 39 , although no good evidence for such activity, or for ADE of ZIKV via prior DENV infection 40 , has emerged from studies of natural infections in humans 41 . Nevertheless, a poorly designed vaccine may (even if natural infections do not) provide opportunities for ADE. Conceivably, given recent insights, regulators may require mutation of the fusion loop region 39 and/or the prM cleavage site 42 to obviate the risk of ADE. Such modifications could be readily incorporated into recombinant vaccines such as SCV-ZIKA/ CHIK, but might be less straightforward for live attenuated or inactivated whole-virus ZIKV vaccines (where viral replication is required in the vaccine recipient or during manufacture). The assay systems illustrating the absence of ADE that would satisfy regulators remain unclear, with in vitro assays perhaps now viewed as less reliable 41 , given inter alia the widespread ability to show ADE in vitro for a range of different viral genera 43 . We have established an IFNAR1 −/− mouse model that shows ADE of DENV disease and were able to show that SCV-ZIKA/CHIK vaccination did not enhance DENV disease in this model ( Supplementary Fig. 5). Although encouraging, the requirements for ZIKV vaccines in this space will have to await vaccine studies in pregnant NHPs and the development of consensus views by regulators.
Although all CHIKV strains are thought to belong to the same serogroup 44 , differences in cross neutralisation between isolates from different genotypes have been reported 45 . The data presented herein suggests increased time post-vaccination increases the cross-neutralising capacity against multiple CHIKV genotypes. Although many neutralising responses are directed at tertiary or quaternary structures 46,47 , a linear epitope (E2EP3) at the N terminus of the E2 protein was identified as the target of many early neutralising responses in CHIKV-infected patients and NHPs 32 . The sequence of this epitope is identical in the CHIKV strain encoded by the SCV vaccine (IOL CHIKV isolate 06-021), the IOL isolate (LR2006-OPY1) and the WA isolate (37997), whereas it has 1 or 2 non-conservative substitutions in the Asian isolates (R99659 and AF15561) (Supplementary Fig. 6). Antibody responses present later in CHIKV infections were found to be less focused on this epitope 32 , suggesting a level of epitope spreading over time. Early E2EP3-focused responses may explain why on day 35   SCV-ZIKA/CHIK induced higher neutralising responses to the IOL and WA isolates (conserved E2EP3 sequences). Later (day 70), epitope spreading may ensure similar levels of cross-neutralising responses to all the genotypes (Fig. 3a, b Mean). Similar broadening of responses has been shown to be promoted by adjuvanting of influenza vaccines 48,49 . The development of broadly neutralising antibodies over several years has also been reported in HIV patients 50 . The data presented herein is a clear illustration of time-dependence for the post-vaccination development of broadly neutralising responses against different CHIKV genotypes.
Precisely, what level of neutralising antibody response is needed to protect against CHIKV is not entirely clear, although a conservative correlate of protection was defined as a reciprocal 50% neutralising serum antibody titre of a ≥10 in a phase 3 trial of a vaccine against the related Ross River virus 51 . In a rapidly lethal interferon signalling-deficient A129 mouse model, a minimum reciprocal 50% neutralising antibody titre of 35 conferred complete protection against CHIKV challenge 52 . After a single vaccination of NHPs herein with 10 7 pfu of SCV-ZIKA/CHIK, the mean day 70 reciprocal FRNT 50 anti-CHIKV titre was 59.4 ± SEM 8.9 (range 28-93) (n = 20; 5 NHPs and 4 CHIKV strains). After two vaccinations with 10 8 pfu, this value was 736 ± SEM 187 (range 90-3090). SCV-ZIKA/CHIK vaccination thus induced neutralising antibody responses to CHIKV that might be expected to provide protection against CHIKV challenge.
The mean SCV-induced anti-VACV reciprocal PRNT 50 neutralisation titre of 79 ± SEM 24 after 2 vaccinations i.m. with 10 8 pfu of SCV vaccines compares favourably with phase III human trial data (i) where MVA vaccination resulted in a mean reciprocal PRNT 50 neutralisation titre of 94 (95% confidence interval (95% CI) 78-113) (n = 189-220) on day 84 after 2 subcutaneous (s.c.) vaccinations (given weeks 0 and 4) with 10 8 TCID 50 of MVA and (ii) where vaccination with the live VACV vaccine, ACAM2000, produced a mean reciprocal PRNT 50 neutralisation titre of 65.6 (95% CI 54-79) (n = 189-220) on day 56 after one vaccination by killing 8 . Although assay systems might differ, both herein and in the study by Pittman et al. 8 , the Western Reserve strain of VACV was used for the antibody assays. The immune correlates of protection against smallpox are not well characterised, with both CD8 T cells and neutralising antibodies thought to play a role 53 . Smallpox (and VACV) exists in two forms: intracellular mature virions (MVs) with one envelope and extracellular enveloped viruses that have an additional envelope. Antibodies directed at both virion forms are believed to be required for optimal protection 54,55 ; however, the VACV used for neutralisation assays usually comprises primarily MVs. The results in Fig. 4 thus suggest that SCV may find utility as a smallpox vaccine; however, NHP challenge studies using monkeypox virus are needed to support this contention.
A number of limitations of this study are recognised. The limited available budget precluded a more extensive dose ranging and scheduling series for the SCV-ZIKA/CHIK vaccine, with only one arguably low end (one vaccination with 10 7 ) and one high end (two vaccination with 10 8 pfu) dosing/scheduling tested. In addition, although poxvirus vaccine systems generally show considerable utility for induction of T-cell responses 1 , these were not accessed in this study, with both CD4 and CD8 T cells likely mediating protection against ZIKV 28,29 . No CHIKV challenge was undertaken due to the high costs associated with NHP studies under BSL3 conditions; furthermore, NHP models are currently also unable to provide arthralgia or overt arthritis readouts 56,57 . Nevertheless, CHIKV neutralising antibody titres were reached that might be expected to provide protection from vireamia and athropathy. We were also unable to gain access to any Brazilian ECSA isolates 58 for use in this industry-supported project; however, such isolates show lower levels of divergence from the vaccine strain than the Asian isolates (to which neutralising response were generated).
In summary, the results of this NHP study of the SCV technology support the view that SCV-ZIKA/CHIK vaccination can induce immunity in NHPs that is sufficient for protection against ZIKV and CHIKV. Perhaps most encouraging was the ability of a single vaccination and a homologous prime-boost to induce significant anti-ZIKV and anti-CHIKV-neutralising antibody responses in NHPs, potentially obviating the need for heterologous prime-boost strategies for recombinant SCV vaccines.

Care, use of animals and AEC approval
The study design was reviewed by the Institutional Animal Care and Use Committee at Southern Research (Internal Approval #18-03-008 F; AAALAC Accreditation #000643; OLAW Assurance D16-00025-Legacy #A3046-1). All animals were cared for and procedures performed in accordance with the institutional guidelines for the care and use of experimental animals, abiding ethical regulations for animal testing and research. Animals were socially housed during the quarantine and prestudy phases, then single-housed following ZIKV challenge. Animals were housed in stainless steel cages that meet requirements set forth in the Animal Welfare Act (Public Law 99-198, USA) and the Guide for the Care and Use of Laboratory Animals (8th Edition, Institute of Animal Resources, Commission on Life Sciences, National Research Council, National Academy Press, Washington DC, 2011). Animals were housed in environmentally monitored and ventilated rooms. Fluorescent lighting provided illumination approximately 12 h per day. Indian rhesus macaques M. mulatta were supplied by PrimGen (Hines, IL, USA), were aged between 1 and 6 years, and weighed between 2.0 and 8.0 kg. A total of 16 animals (8 females and 8 males) were used. All animals were placed into quarantine in the ABSL-2 facility for a minimum of 30 days prior to ZIKV challenge. All animals were screened and confirmed to be free of antibodies to simian immunodeficiency virus, simian retroviruses, simian T-cell leukaemia virus type 1 and Herpes B virus. Animals were also tested and confirmed to be negative for tuberculosis, klebsiella, Trypanosoma cruzi, West Nile virus, DENV and ZIKV.
Macaques were fed twice per day with Purina LabDiet 5048 Certified NHP Diet during the quarantine and study periods. Analyses of the feed, provided by the manufacturer were reviewed by the veterinarian to ensure that no known contaminants were present that could interfere with, or affect, the outcome of the study. In addition, as part of the normal diet, animals were given a variety of fruit and vegetables. Animals were monitored for decreased appetite and/or significant weight loss. All animals were given a unique identification number via a tattoo and were observed twice daily throughout the quarantine and study periods for signs of morbidity and mortality. During the challenge period (day 70-84), animals were observed twice daily for responsiveness and clinical signs including rash, erythema, conjunctivitis, ocular discharge and swelling. Rectal temperatures and body weights of each animal were measured prior to blood collection.

Vaccination and challenge
Prior to study initiation, Indian Rhesus macaques were randomised into respective groups according to gender and weight using Provantis Software (Instem, USA). SCV-ZIKA/CHIK 10 7 pfu had two males and three females; SCV-ZIKA/CHIK 2 × 10 8 pfu had three males and two females; SCVcontrol had two males and two females; inactivated PRVBC59 had one male and one female. Prior to vaccination, bleeding and challenge, animals were anaesthetized using ketamine hydrochloride administered i.m. at 5-30 mg/kg in a volume of 1 ml or less per site. Vaccines were administered i.m. into the right quadriceps (0.5 ml single site), with animals receiving SCV-ZIKA/CHIK or SCV-control (or a positive control formalin-inactivated reference PRVABC59 vaccine 26 ).
Animals were challenged s.c. (anterior surface of the left forearm) with 0.5 ml of wild-type ZIKV strain PRVABC59 (10 5 pfu per animal). Prior to all injections, the injection site was clipped, wiped with alcohol and marked with an indelible marker. Bloods were collected into Serum Separator tubes (2-8 ml) and serum was aliquoted and stored at −80°C.
The SCV-ZIKA/CHIK and SCV-control vaccines were produced in a non-GMP BSL2 SCS line (comprising CHO-S cells transfected with D13L and CP77 13 ) using serum and protein-free cell culture conditions. The vaccines were purified by centrifugation through a sucrose cushion. Briefly, infected cells were harvested by centrifugation. Cell-associated virus was released using multiple freeze-thaw cycles in 10 mM Tris HCl pH 8.0 and 150 mM NaCl. Viral extracts were centrifuged to remove the majority of cell debris. The clarified extract was further purified by centrifugation through a 36% sucrose cushion. The viral pellets were resuspended in 10 mM Tris HCl pH 8, 150 mM NaCl buffer and stored frozen at −80°C.
PCR analyses confirmed the presence of the CHIK and the ZIKA expression cassettes and absence of wild-type SCV 14

Serum viral load determination by qRT-PCR
qRT-PCR was performed using RNA isolated from serum. Briefly, RNA was extracted from serum samples using QIAmp Viral RNA Mini kit (Qiagen, 52906, Germantown, MD, USA) and analysed in triplicate by qRT-PCR using primers directed to the envelope protein (forward primer 5′-TGAGGCAT-CAATATCAGACATG-3′ and reverse primer 5′-GTTCTTTTGCAGACATATT-GAGTG-3′). Five microlitres of purified RNA from each sample was used in a 20 µL qRT-PCR reaction consisting of Fast Virus 4× Master Mix (Applied Biosystems, 4444436, Foster City, CA, USA) containing 500 nM forward and reverse primers with a 200 nM probe. Cycling parameters include the following: an initial reverse transcription step for 5 min at 53°C, followed by 1 min at 95°C and 45 cycles of two-step cycling at 95°C for 5 s and 60°C for 50 s. Data were expressed as viral genome copies/ml using a standard curve established using quantified in vitro-transcribed viral RNA. The LLoRQ was 860 viral genome copies/ml of serum. The LLoRQ represents the lowest limit of 100% reliable quantification over a large number of qRT-PCR experiments using serum samples spiked with in vitro-transcribed ZIKV RNA. CHIKV 50% Focus Reduction Neutralisation Test (FRNT 50 ) NHP serum samples were sent frozen on dry ice to University of Colorado School of Medicine and were thawed and heat inactivated. A FRNT 50 assay was used as described by Hawman et al. 59 . NHP sera were serially diluted in duplicate in Dulbecco's modified Eagle's medium (DMEM)/F:12 medium (Gibco) plus 2% fetal bovine serum (FBS) in 96-well plates (2-fold serial dilutions starting at 1 : 8 dilution). Approximately 100 focus-forming units of the indicated CHIKV isolate were added to each well and the serum plus virus mixture was incubated for 1 h at 37°C. The mixtures were then added for 2 h at 37°C to parallel 96-well plates seeded with Vero cells. The mixtures were then removed and cells were overlaid with 0.5% methylcellulose in MEM/5 % FBS and incubated 18 h at 37°C. Cells were then fixed with 1% paraformaldehyde and probed with 500 ng/ml of the anti-CHIKV monoclonal antibody, CHK-11 60 in wash buffer (1× phosphatebuffered saline (PBS)/0.1% saponin/0.1% bovine serum albumin) for 2 h at room temperature. After washing, cells were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA), diluted 1 : 2000 for 1.5-2 h at room temperature. After washing, CHIKV-positive foci were visualised with TrueBlue peroxidase substrate (SeraCare, Milford MA, USA) and counted using a CTL Biospot Analyzer and Biospot software. The FRNT 50 titre was calculated relative to a virus only (no NHP anti-serum) control set at 100%, using GraphPad Prism 7 (La Jolla, CA, USA) default nonlinear curve fit constrained between 0 and 100%. CHIKV work was conducted in the BSL3 facility at University of Colorado School of Medicine and was approved by institutional biosafety committee (No 09-003). LR2006

VACV Plaque Reduction Neutralisation test (PRNT 50 ) assays
The ability of vaccine-induced antibodies to neutralise VACV was evaluated in a validated VACV-specific PRNT 50 assay in accordance with Southern Research standard operating procedures. Briefly, serum samples were serially diluted in DMEM containing Glutamax and 2% FBS, and added to an equal volume of a fixed dilution of VACV (Western Reserve strain). The serum-virus mixture was then incubated overnight at 2-8°C. Subsequently, 100 µl of each serum-virus mixture was added, in triplicate, to a fresh 24well plate containing confluent Vero cells and incubated at 37 ± 1°C. VACV pre-incubated with normal monkey or vaccinia immune globulin polyclonal antibody serving as negative and positive controls, respectively. Neutralisation endpoint titres were calculated based on the reciprocal dilution of the test serum that produced 50% plaque reduction compared with the virus control.

VACV ELISA assays
To measure VACV-specific antibody responses, serum samples were analysed using a validated VACV-specific ELISA in accordance with Southern Research Frederick Standard Operating Procedure. Briefly, 96well ELISA plates were coated with 100 µl/well of 0.5 µg/ml purified VACV (Western Reserve strain) antigen in PBS and incubated overnight at 2-8°C. Plates were washed using an automated plate washer (BioTek ELx405, Winooski, VT) and then blocked with 5% non-fat milk in 0.05% PBS Tween 20 (PBST) at 37 ± 1°C. Serum samples were serially diluted in triplicate in 5% non-fat milk in 0.05% PBST and added at 100 µl/well. Specific antibodies were detected using a goat anti-monkey HRP-conjugated IgG (Sigma-Aldrich) as secondary antibody. Plates were developed with 100 µl/ well of ABTS substrate for 15-20 min, stopped with 100 µl of 1% SDS in distilled water and read at 405 nm using a SpectraMax plate reader (Molecular Devices. Sunnyvale, CA). Positive (monkey polyclonal antibody) and negative (normal Serum) control samples were included on each plate.

Statistics
Statistics were performed using IBM SPSS Statistics (version19). The twosided t-test was used if the difference in the variances was <4, skewness was > −2 and kurtosis was <2, where the data was nonparametric and N.A. Prow et al.