Efficacy of a ML336 Derivative Against Venezuelan and Eastern Equine Encephalitis Viruses

Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5 mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48 h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25 mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase

Efficacy of a parainfluenza virus 5 (PIV5)-based H7N9 vaccine in mice and guinea pigs: antibody titer towards HA was not a good indicator for protection.

H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7) and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9) in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP) conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively

Polymerase discordance in novel swine influenza H3N2v constellations is tolerated in swine but not human respiratory epithelial cells.

Swine-origin H3N2v, a variant of H3N2 influenza virus, is a concern for novel reassortment with circulating pandemic H1N1 influenza virus (H1N1pdm09) in swine because this can lead to the emergence of a novel pandemic virus. In this study, the reassortment prevalence of H3N2v with H1N1pdm09 was determined in swine cells. Reassortants evaluated showed that the H1N1pdm09 polymerase (PA) segment occurred within swine H3N2 with ∼ 80% frequency. The swine H3N2-human H1N1pdm09 PA reassortant (swH3N2-huPA) showed enhanced replication in swine cells, and was the dominant gene constellation. Ferrets infected with swH3N2-huPA had increased lung pathogenicity compared to parent viruses; however, swH3N2-huPA replication in normal human bronchoepithelial cells was attenuated - a feature linked to expression of IFN-β and IFN-λ genes in human but not swine cells. These findings indicate that emergence of novel H3N2v influenza constellations require more than changes in the viral polymerase complex to overcome barriers to cross-species transmission. Additionally, these findings reveal that while the ferret model is highly informative for influenza studies, slight differences in pathogenicity may not necessarily be indicative of human outcomes after infection

Protection of guinea pigs against H7N9 challenge.

<p>(A) Scheme of immunization and infection. Guinea pigs were immunized IN with PIV5 or PIV5-H7 (10⁷ PFU), or PIV5-H7 + PIV5-NP (5x10⁶ PFU of each). Six guinea pigs were immunized IM with 512 HAUs of iH7N9. At 21 days post immunization (dpi), guinea pigs were bled and HAI titers were measured. The guinea pigs were then infected IN with 10 ID₅₀ of A/Anhui/1/13 (H7N9). One dpi, one naïve guinea pig was co-housed with each infected guinea pig in a single cage. Nasal washes were obtained from guinea pigs at day 2, 4, 6 and 8 after challenge (day post-challenge). Titers of H7N9 in nasal washes were determined by TCID₅₀ assay. (B—E) Nasal wash titers of individual animals. (F) Mean nasal wash titers (+ SEM). (*<i>P</i><0.05 compared to PIV5-vaccinated; Kruskal-Wallis test) (G) Hemagglutination inhibition titers of individual vaccinated animals. (*P < 0.05; Kruskal-Wallis test) Order and shading of bars matches individual guinea pigs for panels B—E, Symbols match for panels F and G.</p

PIV5-H7 and PIV5-NP protected mice against H7N9 challenge.

<p>BALB/c mice in groups of 9–10 were infected IN with PIV5-H7 at a dose of 10⁶ PFU, 10⁶ PFU of PIV5-NP, 10⁶ PFU of PIV5, or PBS. Mice were rested for 8 weeks and were then challenged with 10 50% lethal doses (LD₅₀) of A/Anhui/1/2013 (H7N9) and monitored for (A) survival and (B) weight loss. (*<i>P</i><0.05, log-Rank, compared to PBS or PIV5)</p