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

Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice

The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus.BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus.Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic

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

Dose response of PIV5-H7.

<p>BALB/c mice in groups of 15 were infected IN with PIV5-H7 at a dose of 10⁴, 10⁵ and 10⁶ PFU, or PBS or intramuscularly injected with 256 HAU of iH7N9. Mice were rested for 3 weeks then challenged with 10 LD₅₀ of A/Anhui/1/2013 (H7N9) and monitored for (A) survival and (B) weight loss. (<i>P</i><0.05, ANOVA, *compared to PBS on days 2–8; †compared to PBS on days 2, 6, and 8; ^‡compared to PBS on day 8 (10⁴ only) (C) Titers of H7N9 in the lungs of mice Day 3 post H7N9 challenge. (<i>P</i><0.05, Kruskal-Wallis)</p

Antibody responses in immunized mice.

<p>96-well plates were coated with inactivated H7N9 and incubated overnight. After blocking, serial dilutions of serum, nasal wash or bronchoalveolar lavage (BAL) samples were added to the coated plates. After wash, alkaline phosphatase-labeled goat anti-mouse IgG (A-C) or IgA (D, E) were added and plates were developed using phosphatase substrates. Optical density (OD) was measured at 405 nm on a Bio-Tek Powerwave XS plate reader.</p

Generation and analysis of PIV5 expressing HA of H7N9 (PIV5-H7).

<p>(A) Schematic of PIV5-H7. The PIV5 genome contains seven known transcriptional units and transcribes eight known viral mRNAs. The V and P mRNAs both originate from the same V/P gene by a RNA editing process called pseudo-templated transcription. Leader and trailer sequences are important for viral RNA synthesis and transcription initiation. (B) Expression of H7 in PIV5-H7-infected cells. MDBK cells were mock infected or infected with PIV5-H7. At 2 dpi, the cells were fixed and stained with anti-PIV5-V/P or anti-H7N9 serum. (C) Growth of PIV5-H7. MDBK cells were infected with PIV5 or PIV5-H7 at a moi of 0.1. The media were collected at the indicated intervals and plaque assays were performed on BHK cells.</p

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