Abstract. Three tetravalent formulations of chimeric dengue (DENVax) viruses containing the pre-membrane and envelope genes of serotypes 1-4 expressed by the attenuated DENV-2 PDK-53 genome were tested for safety, immunogenicity, and efficacy in cynomolgus macaques ( Macaca fascicularis ). Subcutaneous injection of the DENVax formulations was well-tolerated. Low levels of viremia of only one of the four vaccine viruses were detected yet virus neutralizing antibody titers were induced against all four dengue virus serotypes after one or two administrations of vaccine. All animals immunized with the high-dose formulation were protected from viremia, and all immunized animals were completely protected from DENV-3 and DENV-4 challenge. A lower dose of DENVax formulation partially protected animals from DENV-1 or DENV-2 challenge. In contrast, all control animals developed high levels of viremia for multiple days after challenge with DENV 1-4. This study highlights the immunogenicity and efficacy of the tetravalent DENVax formulations in nonhuman primates.*Address correspondence to Jorge E. Osorio, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706. E-mail: osorio@svm.vetmed .wisc.edu †These authors contributed equally to this article. PROTECTION BY A DENGUE VACCINE IN MACAQUESderived (DENV-1 16007, DENV-2 16681, DEN-3 16562, and DENV-4 1036). 21 Virus plaque titration was performed under double agarose overlay in six-well plates of confluent Vero cells as described. 20,22 The second agarose overlay containing neutral red vital stain was added four or seven days after infection, depending on the virus plaque phenotypes. Plaques were counted for three consecutive days after the second agarose overlay.Tetravalent DENVax vaccine formulations. The construction and characterization of the four DENVax viruses has been reported. 21 To complete preclinical development of DENVax, new viral stocks were generated by introducing RNAs transcribed from infectious cDNA clones (pD2-PDK53 and chimeric pD2/1, /3, and /4 plasmids) into the certified vaccine production Vero cells by electroporation as described 21 under Good Manufacturing Practice (GMP) conditions at Shantha Biotechnics. The viruses were amplified, plaque purified, characterized, and sequenced for each of the four DENVax serotypes. On the basis of these analyses, a formal pre-master virus seed was chosen for each DENVax serotype and was then amplified to generate the master virus seed for each serotype (Huang, C. and others, unpublished data).For this study, individual pre-master seed viruses were used to make non-GMP surrogate master seeds in serum-free media as follows. Viruses diluted in DMEM to achieve a multiplicity of infection of 0.001 were adsorbed for 1.5 hours onto rinsed Vero cell monolayers at 37°C. After adsorption, the monolayers were rinsed three times with phosphate-buffered saline, and then fresh serum-free DMEM medium was added. The viruses were grown for 8-12 days in an atmosphere of 5% CO...

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes explosive outbreaks of febrile illness associated with rash, and painful arthralgia. The CHIK vaccine strain 181/clone25 (181/25) developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) was shown to be well-tolerated and highly immunogenic in phase I and II clinical trials although it induced transient arthralgia in some healthy adult volunteers. In an attempt to better understand the host factors that are involved in the attenuating phenotype of CHIK 181/25 vaccine virus we conducted studies in interferon (IFN)-compromised mice and also evaluated its immunogenic potential and protective capacity. Infection of AG129 mice (defective in IFN-α/β and IFN-γ receptor signaling) with CHIK 181/25 resulted in rapid mortality within 3-4 days. In contrast, all infected A129 mice (defective in IFN-α/β receptor signaling) survived with temporary morbidity characterized by ruffled appearance and body weight loss. A129 heterozygote mice that retain partial IFN-α/β receptor signaling activity remained healthy. Infection of A129 mice with CHIK 181/25 induced significant levels of IFN-γ and IL-12 while the inflammatory cytokines, TNFα and IL-6 remained low. A single administration of the CHIK 181/25 vaccine provided both short-term and long-term protection (38 days and 247 days post-prime, respectively) against challenge with wt CHIKV-La Reunion (CHIKV-LR). This protection was at least partially mediated by antibodies since passively transferred immune serum protected both A129 and AG129 mice from wt CHIKV-LR and 181/25 virus challenge. Overall, these data highlight the importance of IFNs in controlling CHIK 181/25 vaccine and demonstrate the ability of this vaccine to elicit neutralizing antibody responses that confer short-and long-term protection against wt CHIKV-LR challenge.

Formulations of chimeric dengue vaccine (DENVax) viruses containing the pre-membrane (prM) and envelope (E) genes of serotypes 1–4 expressed in the context of the attenuated DENV-2 PDK-53 genome were tested for safety, immunogenicity and efficacy in interferon receptor knock-out mice (AG129). Monovalent formulations were safe and elicited robust neutralizing antibody responses to the homologous virus and only limited cross-reactivity to other serotypes. A single dose of monovalent DENVax-1, -2, or -3 vaccine provided eighty or greater percent protection against both wild-type (wt) DENV-1 (Mochizuki strain) and DENV-2 (New Guinea C strain) challenge viruses. A single dose of monovalent DENVax-4 also provided complete protection against wt DENV-1 challenge and significantly increased the survival times after challenge with wt DENV-2. In studies using tetravalent mixtures, DENVax ratios were identified that: (i) caused limited viremia, (ii) induced serotype-specific neutralizing antibodies to all four DENV serotypes with different hierarchies, and (iii) conferred full protection against clinical signs of disease following challenge with either wt DENV-1 or DENV-2 viruses. Overall, these data highlight the immunogenic profile of DENVax, a novel candidate tetravalent dengue vaccine and the advantage of sharing a common attenuated genomic backbone among the DENVax monovalent vaccines that confer protection against homologous or heterologous virus challenge.

Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus IMPORTANCECurrent influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging pandemic viruses. Therefore, we evaluated a vaccine strategy designed to induce both antibody and T cell responses, which may provide more broadly cross-protective immunity against influenza. Here, we show in a translational primate model that vaccination with a modified vaccinia virus Ankara encoding hemagglutinin from a heterosubtypic H5N1 virus was associated with reduced shedding of a pandemic H1N1 virus challenge, while vaccination with MVA encoding nucleoprotein, an internal viral protein, was not. Unexpectedly, this reduced shedding was associated with nonneutralizing antibodies that bound H1 hemagglutinin and activated natural killer cells. Therefore, antibody-dependent cellular cytotoxicity (ADCC) may play a role in cross-protective immunity to influenza virus. Vaccines that stimulate ADCC antibodies may enhance protection against pandemic influenza virus.

Development of an influenza vaccine that provides cross-protective immunity remains a challenge. Candidate vaccines based on a recombinant modified vaccinia Ankara (MVA) viral vector expressing antigens from influenza (MVA/Flu) viruses were constructed. A vaccine candidate, designated MVA/HA1/C13L/NP, that expresses the hemagglutinin from pandemic H1N1 (A/California/04/09) and the nucleoprotein (N1) from highly pathogenic H5N1 (A/Vietnam/1203/04) fused to a secretory signal sequence from vaccinia virus was highly protective. The vaccine elicited strong antibody titers to homologous H1N1 viruses while cross-reactive antibodies to heterologous viruses were not detectable. In mice, this MVA/HA1/C13L/NP vaccine conferred complete protection against lethal challenge with A/Vietnam/1203/04 (H5N1), A/Norway/3487-2/09 (pandemic H1N1) or A/Influenza/Puerto Rico/8/34 (seasonal H1N1) and partial protection (57.1%) against challenge with seasonal H3N2 virus (A/Aichi/68). The protective efficacy of the vaccine was not affected by pre-existing immunity to vaccinia. Our findings highlight MVA as suitable vector to express multiple influenza antigens that could afford broad cross-protective immunity against multiple subtypes of influenza virus.