Darwyn Kobasa

Lethal Influenza Virus Infection in Macaques Is Associated with Early Dysregulation of Inflammatory Related Genes

The enormous toll on human life during the 1918–1919 Spanish influenza pandemic is a constant reminder of the potential lethality of influenza viruses. With the declaration by the World Health Organization of a new H1N1 influenza virus pandemic, and with continued human cases of highly pathogenic H5N1 avian influenza virus infection, a better understanding of the host response to highly pathogenic influenza viruses is essential. To this end, we compared pathology and global gene expression profiles in bronchial tissue from macaques infected with either the reconstructed 1918 pandemic virus or the highly pathogenic avian H5N1 virus A/Vietnam/1203/04. Severe pathology was observed in respiratory tissues from 1918 virus-infected animals as early as 12 hours after infection, and pathology steadily increased at later time points. Although tissues from animals infected with A/Vietnam/1203/04 also showed clear signs of pathology early on, less pathology was observed at later time points, and there was evidence of tissue repair. Global transcriptional profiles revealed that specific groups of genes associated with inflammation and cell death were up-regulated in bronchial tissues from animals infected with the 1918 virus but down-regulated in animals infected with A/Vietnam/1203/04. Importantly, the 1918 virus up-regulated key components of the inflammasome, NLRP3 and IL-1β, whereas these genes were down-regulated by A/Vietnam/1203/04 early after infection. TUNEL assays revealed that both viruses elicited an apoptotic response in lungs and bronchi, although the response occurred earlier during 1918 virus infection. Our findings suggest that the severity of disease in 1918 virus-infected macaques is a consequence of the early up-regulation of cell death and inflammatory related genes, in which additive or synergistic effects likely dictate the severity of tissue damage.

Assessment of the Efficacy of Commercially Available and Candidate Vaccines against a Pandemic H1N1 2009 Virus

Abstract Background. The emergence and global spread of the pandemic H1N1 2009 influenza virus have raised questions regarding the protective effect of available seasonal vaccines and the efficacy of a newly produced matched vaccine. Methods. Ferrets were immunized with the 2008–2009 formulations of commercially available live attenuated (FluMist; MedImmune) or split-inactivated (Fluviral; GlaxoSmithKline) vaccines, a commercial swine vaccine (FluSure; Pfizer), or a laboratory-produced matched inactivated whole-virus vaccine (A/Mexico/InDRE4487/2009). Adaptive immune responses were monitored, and the animals were challenged with A/Mexico/InDRE4487/2009 after 5 weeks. Results. Only animals that received the swine or matched vaccines developed detectable hemagglutination- inhibiting antibodies against the challenge virus, whereas a T cell response was exclusively detected in animals vaccinated with FluMist. After challenge, all animals had high levels of virus replication in the upper respiratory tract. However, preexisting anti—pandemic H1N1 2009 antibodies resulted in reduced clinical signs and improved survival. Surprisingly, FluMist was associated with a slight increase in mortality and greater lung damage, which correlated with early up-regulation of interleukin-10. Conclusions. The present study demonstrates that a single dose of matched inactivated vaccine confers partial protection against a pandemic H1N1 2009 virus, and it suggests that a higher dose or prime-boost regimen may be required. The consequences of mismatched immunity to influenza merit further investigation.

Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1 viruses

The combination of rapid evolution and high mortality in human cases of infections has raised concerns that the H5N1 avian influenza virus may become a new, possibly severe, pandemic virus. Vaccination is likely to be the most efficient strategy to mitigate the impact of the next influenza pandemic. The present study evaluates B and T cell immune responses generated by the H5N1 viral antigens, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), or the M2 ion channel in parallel, expressed from a DNA vaccine vehicle. Protection studies of immunized mice challenged with 100 LD50 of homologous or heterologous H5N1 viruses indicate that HA afforded better protection than the NA, NP or M2 DNA vaccines. The antibody response was also higher in HA-vaccinated mice as determined by hemagglutination inhibition (HI) and neutralizing antibodies (NAB) assays. Interestingly, the T cell response was higher against HA than against NA, NP or M2 and was detectable at low doses of the DNA-HA vaccine capable of inducing complete protection, despite the absence of a detectable B cell response. This study emphasizes the need to evaluate the relationship between both arms of the adaptive immune responses in regards to protective efficacy against influenza virus.

A (H1N1) pdm09 HA D222 variants associated with severity and mortality in patients during a second wave in Mexico

BackgroundPandemic type A (H1N1) influenza arose in early 2009, probably in Mexico and the United States, and reappeared in North America in September for seven more months. An amino acid substitution in the hemagglutinin (HA), D222G, has been reported in a significant proportion of patients with a severe and fatal outcome. We studied the prevalence of HA222 substitutions in patients in Mexico during the second wave and its association with clinical outcome and pathogenicity in a mouse model.MethodsThe nucleotide sequences of hemagglutinin (HA) from viruses collected from 77 patients were determined including 50 severe and fatal cases and 27 ambulatory cases. Deep sequencing was done on 5 samples from severe or fatal cases in order to determine the quasispecies proportion. Weight loss and mortality due to infection with cultured influenza viruses were analyzed in a mouse model.ResultsViruses from 14 out of 50 hospitalized patients (28%) had a non aspartic acid residue at the HA 222 position (nD222), while all 27 ambulatory patients had D222 (p = 0.0014). G222 was detected as sole species or in coexistence with N222 and D222 in 12 patients with severe disease including 8 who died. N222 in coexistence with D222 was detected in 1 patient who died and co-occurrence of A222 and V222, together with D222, was detected in another patient who died. The patients with a nD222 residue had higher mortality (71.4%), compared to the group with only D222 (22.2%, p = 0.0008). Four of the 14 viruses from hospitalized patients were cultured and intranasally infected into mice. Two viruses with G222 were lethal while a third virus, with G222, caused only mild illness in mice similar to the fourth virus that contained D222.ConclusionsWe confirm the elevated incidence of HA222 (H1N1)pdm09 variants in severe disease and mortality. Both clinical and mouse infection data support the idea that nD222 mutations contribute to increased severity of disease but additional determinants in disease outcome may be present.

Development and Characterization of a Guinea Pig-Adapted Sudan Virus

ABSTRACT Infections with Sudan virus (SUDV), a member of the genus Ebolavirus, result in a severe hemorrhagic fever with a fatal outcome in over 50% of human cases. The paucity of prophylactics and therapeutics against SUDV is attributed to the lack of a small-animal model to screen promising compounds. By repeatedly passaging SUDV within the livers and spleens of guinea pigs in vivo, a guinea pig-adapted SUDV variant (SUDV-GA) uniformly lethal to these animals, with a 50% lethal dose (LD50) of 5.3 × 10−2 50% tissue culture infective doses (TCID50), was developed. Animals infected with SUDV-GA developed high viremia and died between 9 and 14 days postinfection. Several hallmarks of SUDV infection, including lymphadenopathy, increased liver enzyme activities, and coagulation abnormalities, were observed. Virological analyses and gross pathology, histopathology, and immunohistochemistry findings indicate that SUDV-GA replicates in the livers and spleens of infected animals similarly to SUDV infections in nonhuman primates. These developments will accelerate the development of specific medical countermeasures in preparation for a future disease outbreak due to SUDV. IMPORTANCE A disease outbreak due to Ebola virus (EBOV), suspected to have emerged during December 2013 in Guinea, with over 11,000 dead and 28,000 infected, is finally winding down. Experimental EBOV vaccines and treatments were administered to patients under compassionate circumstances with promising results, and availability of an approved countermeasure appears to be close. However, the same range of experimental candidates against a potential disease outbreak caused by other members of the genus Ebolavirus, such as Sudan virus (SUDV), is not readily available. One bottleneck contributing to this situation is the lack of a small-animal model to screen promising drugs in an efficient and economical manner. To address this, we have generated a SUDV variant (SUDV-GA) that is uniformly lethal to guinea pigs. Animals infected with SUDV-GA develop disease similar to that of SUDV-infected humans and monkeys. We believe that this model will significantly accelerate the development of life-saving measures against SUDV infections.

Recently Emerged Swine Influenza A Virus (H2N3) Causes Severe Pneumonia in Cynomolgus Macaques

The triple reassortant H2N3 virus isolated from diseased pigs in the United States in 2006 is pathogenic for certain mammals without prior adaptation and transmits among swine and ferrets. Adaptation, in the H2 hemagglutinin derived from an avian virus, includes the ability to bind to the mammalian receptor, a significant prerequisite for infection of mammals, in particular humans, which poses a big concern for public health. Here we investigated the pathogenic potential of swine H2N3 in Cynomolgus macaques, a surrogate model for human influenza infection. In contrast to human H2N2 virus, which served as a control and largely caused mild pneumonia similar to seasonal influenza A viruses, the swine H2N3 virus was more pathogenic causing severe pneumonia in nonhuman primates. Both viruses replicated in the entire respiratory tract, but only swine H2N3 could be isolated from lung tissue on day 6 post infection. All animals cleared the infection whereas swine H2N3 infected macaques still presented with pathologic changes indicative of chronic pneumonia at day 14 post infection. Swine H2N3 virus was also detected to significantly higher titers in nasal and oral swabs indicating the potential for animal-to-animal transmission. Plasma levels of IL-6, IL-8, MCP-1 and IFNγ were significantly increased in swine H2N3 compared to human H2N2 infected animals supporting the previously published notion of increased IL-6 levels being a potential marker for severe influenza infections. In conclusion, the swine H2N3 virus represents a threat to humans with the potential for causing a larger outbreak in a non-immune or partially immune population. Furthermore, surveillance efforts in farmed pig populations need to become an integral part of any epidemic and pandemic influenza preparedness.

Co-administration of certain DNA vaccine combinations expressing different H5N1 influenza virus antigens can be beneficial or detrimental to immune protection.

Achieving broad-spectrum immunity against emerging zoonotic viruses such as avian influenza H5N1 and other possible pandemic viruses will require generation of cross-protective immune responses. Strong antibody responses generated against the H5HA protein are protective, however, antigenic variation between diverging isolates can interfere with virus neutralization. The current study investigates co-administration of an H5 HA DNA vaccine with other variable and conserved influenza antigens (NA, NP, and M2). All antigens were derived from the A/Hanoi/30408/2005 (H5N1) virus and the contribution towards overall protection and immune activation was assessed against lethal homologous and heterologous challenges. An (HA+NA) combination afforded the best protection against homologous challenge and (HA+NP) was comparable to HA alone against heterologous A/Hong Kong/483/1997 challenge. Interestingly, combining all four H5 antigens at a single site did not improve protection against matched challenge and unexpectedly reduced survival by 30% against a heterologous challenge. Survival was also significantly decreased against heterologous challenge following combination of (HA+NP) with an unrelated antigen. Although there were no significant changes in antibody titres, significantly lower T-cell responses were detected against all antigens except HA in each combination. Co-administration of the vaccines at different injection sites restored T-cell responses but did not improve overall protection. Similar observations were also recorded following combination of HA and NP antigens using two different adenovirus-based backbones. Overall, the data suggest that co-administering certain H5N1 antigens offer better or comparable protection to HA alone, however, combining extra antigens may be unnecessary and lead to unfavourable immune responses.

Randomized Controlled Ferret Study to Assess the Direct Impact of 2008–09 Trivalent Inactivated Influenza Vaccine on A(H1N1)pdm09 Disease Risk

During spring-summer 2009, several observational studies from Canada showed increased risk of medically-attended, laboratory-confirmed A(H1N1)pdm09 illness among prior recipients of 2008–09 trivalent inactivated influenza vaccine (TIV). Explanatory hypotheses included direct and indirect vaccine effects. In a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008–09 TIV may have directly influenced A(H1N1)pdm09 illness. Thirty-two ferrets (16/group) received 0.5 mL intra-muscular injections of the Canadian-manufactured, commercially-available, non-adjuvanted, split 2008–09 Fluviral or PBS placebo on days 0 and 28. On day 49 all animals were challenged (Ch0) with A(H1N1)pdm09. Four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (Ch+5) and the rest followed until Ch+14. Sera were tested for antibody to vaccine antigens and A(H1N1)pdm09 by hemagglutination inhibition (HI), microneutralization (MN), nucleoprotein-based ELISA and HA1-based microarray assays. Clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. Baseline characteristics were similar between the two groups of influenza-naïve animals. Antibody rise to vaccine antigens was evident by ELISA and HA1-based microarray but not by HI or MN assays; virus challenge raised antibody to A(H1N1)pdm09 by all assays in both groups. Beginning at Ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at Ch+5 (7.4% vs. 5.2%; p = 0.01). At Ch+5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/mL, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p>0.05). At Ch+14, both groups had recovered. Findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. While they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008–09 TIV receipt on A(H1N1)pdm09 illness. As such, they warrant further in-depth investigation and search for possible mechanistic explanations.

Prior infection of chickens with H1N1 avian influenza virus elicits heterologous protection against highly pathogenic H5N2

Current vaccines for influenza are primarily killed whole virus vaccines that elicit antibody responses to the homologous virus but lack protection against heterologous viruses. Using chickens as a model we have explored the possibility of using a live low pathogenic avian influenza (LPAI) A/goose/AB/223/2005 H1N1 virus as a vaccine to generate protective immunity against heterologous highly pathogenic avian influenza (HPAI) A/chicken/Pensylvania/1370/1983 H5N2 virus challenge. Virus replicated in chickens infected with LPAI H1N1 but did not cause clinical disease. In addition, these chickens developed neutralizing antibodies to LPAI H1N1 virus, but not HPAI H5N2, 21 days post infection (DPI). Furthermore, peripheral blood mononuclear cells from H1N1-infected chickens at 20 DPI had antigen specific proliferation and IFN-γ secretion following antigen stimulation to H5N2 indicating a heterologous HPAI H5N2 specific cell mediated immunity (CMI) following LPAI H1N1 infection. Following challenge with HPAI H5N2 virus, all control chickens developed clinical disease, while chickens previously infected with H1N1 did not develop clinical disease and shed significantly less virus by oral and cloacal routes. These results indicated that previous infection with LPAI virus can generate heterologous CMI capable of protecting against HPAI H5N2.

Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry

For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P2-positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P2 production in cells to promote efficient delivery to NPC1.