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We studied the replication of influenza A/California/07/09 (H1N1) wild type (CA09wt) virus in two non-human primate species and used one of these models to evaluate the immunogenicity and protective efficacy of a live attenuated cold-adapted vaccine, which contains the hemagglutinin and neuraminidase from the H1N1 wild type virus and six internal protein gene segments of the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. We infected African green monkeys (AGMs) and rhesus macaques with 2 × 106 TCID50 of CA09wt and CA09ca influenza viruses. The virus replicated in the upper respiratory tract of all animals but the titers in upper respiratory tract tissues of rhesus macaques were significant higher than in AGMs (mean peak titers 104.5 TCID50/g and 102.0 TCID50/g on days 4 and 2 post-infection, respectively; p<0.01). Virus replication was observed in the lungs of all rhesus macaques (102.0–105.4TCID50/g) whereas only 2 out of 4 AGMs had virus recovered from the lungs (102.5– 103.5 TCID50/g). The CA09ca vaccine virus was attenuated and highly restricted in replication in both AGMs and rhesus macaques. We evaluated the immunogenicity and protective efficacy of the CA09ca vaccine in rhesus macaques because CA09wt virus replicated more efficiently in this species. One or two doses of vaccine were administered intranasally and intratracheally to rhesus macaques. For the two-dose group, the vaccine was administered 4-weeks apart. Immunogenicity was assessed by measuring hemagglutination-inhibiting (HAI) antibodies in the serum and specific IgA antibodies to CA09wt virus in the nasal wash. One or two doses of the vaccine elicited a significant rise in HAI titers (range 40–320). Two doses of CA09ca elicited higher pH1N1-specific IgA titers than in the mock-immunized group (p<0.01). Vaccine efficacy was assessed by comparing titers of CA09wt challenge virus in the respiratory tract of mock immunized and CA09ca vaccinated monkeys. Significantly lower virus titers were observed in the lungs of vaccinated animals than mock-immunized animals (p ≤ 0.01). Our results demonstrate that AGMs and rhesus macaques support the replication of pandemic H1N1 influenza virus to different degrees and a cold-adapted pH1N1 vaccine elicits protective immunity against pH1N1 virus infection in rhesus macaques.
The 2009 swine-origin H1N1 influenza virus caused the first influenza pandemic of the 21st century [1–3]. We had previously studied the pathogenesis of pH1N1 (A/California/7/2009 H1N1, CA09wt)  and evaluated the efficacy and immunogenicity of a live attenuated cold adapted (ca) reassortant vaccine, A/California/7/2009 (CA09ca), in mice and ferrets . Although small animal models such as mice and ferrets are often used to study the pathogenesis of influenza viruses, these animals do not fully mirror the response to live attenuated influenza vaccines in humans [6, 7]. Non-human primate models have been used less extensively in influenza vaccine research. New World [squirrel monkeys (Saimiri sciureus), owl monkeys (Aotus trivirgatus) and cebus monkeys (Cebus apella and Cebus albifrons)] and Old World species of monkeys [pigtailed macaques (Macaca nemestrina) and Cynomolgus macaques (Macaca fascicularis)] have been evaluated as models for influenza virus infection and a broad spectrum of clinical signs and degree of influenza virus replication in the respiratory tract were observed among the different species [8–13]. Squirrel monkeys developed mild upper respiratory illness following intratracheally inoculation of influenza A/Udorn/72 (H3N2), A/Alaska/77 (H3N2) and A/Hong Kong/77 (H1N1) viruses whereas no clinical signs were observed in owl and cebus monkeys infected with these viruses . Among the Old World monkeys, pigtailed macaques (Macaca nemestrina) exhibited clinical signs of infection such as weight loss, nasal discharge and moderate fever following inoculation of A/Texas/91 (H1N1) by multiple routes . Cynomolgus macaques (Macaca fascicularis) have been used to study highly pathogenic H5N1 viruses [14, 15], the 1918 pandemic virus . Thus far, three published studies have investigated the pathogenic potential of the pH1N1 virus in cynomolgus macaques. pH1N1 virus-infected cynomolgus macaques showed clinical disease ranging from mild to severe pneumonia that was more severe than the mild illness associated with seasonal H1N1 virus . The pH1N1 virus replicated efficiently in the lungs and other organs of infected cynomolgus macaques whereas seasonal influenza viruses were typically limited in their replicative ability to the lungs of infected primates [11–13]. Pathological examination revealed that pH1N1 infection caused lung lesions that were intermediate in severity between those observed following infection with highly pathogenic avian H5N1 and seasonal H1N1 viruses. Although pathogenesis of the pH1N1 virus has been studied in non-human primates, there are no reports of the evaluation of the pH1N1 virus vaccine in this animal model.
We designed a study to evaluate the immunogenicity and protective efficacy of the live attenuated CA09ca pH1N1 vaccine in non-human primates. We found that replication of the CA09wt virus was different in two species of non-human primates; rhesus macaques supported replication of CA09wt and CA09ca viruses better than African green monkeys. In both species, the CA09wt virus replicated in the upper and lower respiratory tract, whereas replication of the CA09ca vaccine strain was severely restricted in the lower respiratory tract. We studied the immunogenicity and protective efficacy of the CA09ca virus in rhesus macaques, and found that vaccination with either 1 or 2 doses of vaccine elicited a protective antibody titer and conferred protection against challenge with the CA09wt virus.
The wild type (wt) pandemic H1N1 virus, A/California/7/2009 (CA09wt), was kindly provided by the Influenza Division, Centers for Disease Control and Prevention (CDC). The CA09wt virus was propagated in the allantoic cavity of 9-to 11-day-old embryonated specific pathogen-free hen’s eggs. The titer of the virus was determined in Madin-Darby Canine Kidney (MDCK) cells. Allantoic fluid from passage 4 was used in this study. There are 2 amino acid differences (N125D and Q223R) between the HA proteins of the CA09wt virus used in this study and those available in Genbank (FJ969540.1). The live attenuated cold-adapted (ca) reassortant virus, A/California/7/2009 (CA09ca) H1N1 virus, was generated by reverse genetics . In brief, the H1 HA and N1 NA of the reassortant pandemic H1N1 ca vaccine virus were derived from the CA09wt virus, and the internal gene segments were derived from A/Ann Arbor/6/60 ca (AA ca; H2N2) master donor virus. The CA09ca virus was propagated in embryonated eggs and passage 3 was used in this study. The CA09ca virus contains two additional amino acid changes (K119E and A186D) in the HA protein that enhanced vaccine virus yield in eggs, without affecting vaccine antigenicity and immunogenicity in ferrets .
Studies were carried out in 25 approximately 3- to 4-year-old male or female African green monkeys (AGM; Chlorocebus aethiops) and 37 rhesus macaques (Macaca mulatta). The monkeys were examined each day for evidence of clinical illness such as body temperature, pulse, appetite, behavior, stool, respiratory symptoms, and ocular signs. All animal experiments were done at the NIH, in compliance with the guidelines of the NIAID/NIH Animal Care and Use Committee.
For inoculation and immunization, the monkeys were anesthetized by intramuscular injection (I.M.) with Ketamine HCl (10mg/kg) and the wt or vaccine virus was delivered intranasally (I.N.) and intratracheally (I.T.) with 1 ml by each route containing 1 × 106 TCID50 of the virus. Four animals in each group received 1 or 2 doses of vaccine. Additionally, two monkeys in each group were mock immunized. Sera were collected on days 28 and 56 following immunization.
The HAI assay was performed as previously described [18, 19]. Briefly, ferret sera treated with receptor destroying enzyme (RDE, SEIKEN, Campbell, CA) were 2-fold serially diluted in 96-well V-bottom plates starting at a dilution of 1:10, and 4 HA units of virus was added. Control wells received PBS alone or PBS with virus in the absence of antibody. Virus and sera were incubated together for 30 min at room temperature. Next, 50 μl of a 0.5% (vol/vol) suspension of turkey erythrocytes was added. The antibody, virus, and erythrocytes were gently mixed, and the results were recorded after incubating for 45–60 min at room temperature. HAI titers were recorded as the inverse of the highest antibody dilution that inhibited hemagglutination.
ELISA was performed as previously described . Briefly, plates were coated with β-propiolactone-inactivated CA09wt virus at 1,000 hemagglutinating units (HAU)/ml (50 μl/well) or baculovirus-expressed recombinant HA protein from the pH1N1 virus (NR15258) obtained through the National Institutes of Health Biodefense and Emerging Infectious Diseases Repository and used at 1 μg/ml (50 μl per well). Nasal wash samples were incubated on the plates at 4°C overnight. Bound antibodies were detected with biotinylated goat anti-monkey IgA (α-chain) and HRP conjugated streptavidin (Dako, Carpinteria, CA). Wells with an OD of >0.2 at 450 nm were considered positive.
To determine the replication kinetics of CA09wt and CA09ca viruses, groups of animals were inoculated with 2 × 106 TCID50 of the respective viruses. On days 2, 4 and 7 after inoculation, animals were euthanized and eight portions of lung (right caudal distal and proximal, left caudal distal and proximal, right cranial distal and proximal, left cranial distal and proximal), nasal turbinates, and intestines (duodenum, ileocecal valve, jejuno-ileal loop and descending colon) were harvested from each group. Organs were homogenized in L-15 medium containing antibiotic-antimycotic (Invitrogen, Carlsbad, CA) to make 10% (weight/volume) tissue homogenates. Tissue homogenates were clarified by centrifugation and titrated in 24- and 96-well tissue culture plates containing MDCK cell monolayers, as described previously . Titers are expressed as log10 TCID50/g tissue.
HAI antibody titers in pre- and post-vaccination serum samples were determined by standard methods and were defined as the reciprocal of highest dilution of serum that completely inhibited the agglutination of erythrocytes by 4 HA units of virus antigen. The HAI titers of each group are presented as geometric mean titers (GMT). CA09 specific IgA titers in nasal wash of pre- and post-vaccination animals were determined by ELISA as described above. An optical density (OD) > 0.2 was considered to be positive.
To determine the efficacy of the CA09ca vaccine, rhesus macaques were challenged with the CA09wt virus and the level of replication of the challenge virus in the respiratory tract of vaccinated animals was compared with the titers in the mock-immunized group. Nasal washes and nasal swabs were collected on days 2 and 4 post-challenge. At necropsy on day 4, tracheal lavage, nasal turbinates, trachea, lungs and intestines were collected. Virus titers were determined as described above.
Single-cell suspensions from lung tissues were prepared as previously described [20, 22]. In brief, the lungs were digested for 15 min at 37°C in media containing 1 mg/ml collagenase (Roche Diagnostics GmbH, Mannheim, Germany) and 0.02 mg/ml DNase (Sigma-Aldrich, St. Louis, MO). The treated lungs were pressed through a cell strainer (BD PharMingen, San Diego, CA) to obtain a single-cell suspension. The cells were stained with FITC-CD3, PE-CD4, APC-CD8, FITC-CD14, APC-CD11c, PE-CD11b (BD PharMingen, San Diego, CA) and Biotinylated-CD68 (Biolegend, San Diego, CA). After fixing with 2% paraformaldehyde, the cells were analyzed on a FACSCalibur (BD Biosciences, San Jose, CA) and FlowJo Software (Tree Star, Inc, Ashland, OR).
The significance of differences between different groups was assessed using the unpaired Student’s t test using Prism 5 (GraphPad Software, Inc. San Diego, CA); p values <0.05 were considered significantly different.
Groups of 12 AGMs and rhesus macaques were inoculated intranasally and intratracheally with 2 × 106 TCID50 of the wild type pH1N1 virus (CA09wt) or the live attenuated vaccine (CA09ca). Four monkeys in each group were euthanized and organs were harvested on days 2, 4 and 7 post-infection (Fig. 1A). None of the AGMs and rhesus macaques that received these viruses developed clinical signs of infection such as increased body temperature, weight loss, fever or signs of respiratory distress. The CA09wt virus replicated in the nasal turbinates and trachea of experimentally inoculated AGMs and rhesus macaques. The CA09wt virus was detected in the nasal turbinates of all 4 animals on day 2 post-infection. The degree of replication of the CA09wt virus in the nasal turbinates and trachea was higher in rhesus macaques than in AGMs. Peak replication in the upper respiratory tract was observed on day 4 post-infection in rhesus macaques, and 2 days post-infection in AGMs (Fig. 1B and 1C). Replication of the CA09ca vaccine virus was severely restricted in the upper respiratory tract; virus was detected in the nasal turbinates of 1 of 4 AGMs and 4 of 4 rhesus macaques 2 days post-infection (Fig. 1B and 1C).
Evidence of replication of the CA09wt and CA09ca viruses was also observed in nasal swabs, nasal washes and tracheal lavages from monkeys on days 2, 4 and 7 post-infection. In AGMs, CA09wt virus was detected in nasal swabs but not from nasal washes and tracheal lavage samples. Virus was detected in nasal swabs of all 4 animals on day 4 post-infection (Fig. 2A). CA09wt virus was detected from nasal swabs, nasal washes and tracheal lavage samples of rhesus macaques; the mean virus titer in respiratory secretion samples from rhesus macaques was slightly higher on day 4 than on day 2 or day 7 post-infection (Fig. 2B). The CA09ca virus was not detected in respiratory secretions of AGMs or rhesus macaques.
The CA09wt virus replicated inconsistently in AGM lungs; viral replication was observed in only 4 AGMs. Replication was observed in all rhesus macaques that received the CA09wt virus and the peak of replication was seen on day 4 post-infection (Table 1). Replication of the CA09ca virus was not detected in the lungs of infected AGMs or rhesus macaques (data not shown) consistent with the temperature sensitive phenotype of the vaccine virus .
Interestingly, replication of CA09wt virus was observed by virus titration in the jejuno-ileal (1/12; 2.95 TCID50/g) and ileocecal valve (2/12; 3.7 and 2.5 TCID50/g) region of the small intestine of rhesus macaques on days 4 or 7 post-infection but not in AGMs.
To determine the cellular composition in lung tissues, single-cell suspensions were prepared and cell populations were detected using cellular makers. We determined the number of CD4 T cells (CD4+CD3+), CD8 T cells (CD8+ CD3+) and macrophages (CD11b+CD11c-CD68+) in the lungs of CA09wt and CA09ca virus infected AGMs (Fig. 3A) and rhesus macaques (Fig. 3B) on days 2, 4 and 7 post-infection. The number of CD4+ and CD8+ T cells in the lungs of infected animals remained stable from days 2 to 7 post-infection. Interestingly, an increased number of macrophages were observed in the lungs of CA09wt virus infected AGMs (Fig. 3A left panel) and rhesus macaques (Fig. 3B left panel) but not in animals that received the CA09ca vaccine virus (Fig. 3A and 3B right panel).
We used rhesus macaques to study the immunogenicity and efficacy of the CA09ca vaccine because the CA09wt virus replicated more efficiently in this species (Fig. 1 and Table 1). Rhesus macaques were immunized with either 1 or 2 doses of vaccine (4 animals per group) (Fig. 4A). An additional 2 monkeys in each group were mock immunized with L-15. Four weeks post-vaccination, the animals that received 1 dose of the vaccine were challenged with the 2x106 TCID50/ml of CA09wt virus, which was previously shown to result in 100% infection in rhesus macaque (Table 1.), and the animals that received 2 doses of vaccine were challenged 4 weeks after the second dose of vaccine. pH1N1-specific IgA antibody was detected in nasal wash samples on days 28 and 56 post-immunization (Fig. 4B). Animals that received 2 doses of vaccine had higher pH1N1-specific IgA titers than animals that received one dose of vaccine. Serum samples were collected on days 0, 28 and 56 post-immunization. On day 28, after the first dose of vaccine, all animals developed hemagglutination inhibition (HAI) antibodies in their sera, with titers ranging from 1:40 to 1:80 (Table 2). After the second dose of vaccine, the titer of HAI antibody was significantly higher (ranging from 160 to 320, Table 2). HAI antibodies were not detected in mock-immunized animals.
The protective efficacy of the CA09ca vaccine against homologous CA09wt virus challenge was evaluated in respiratory tissues and secretions on day 4 after challenge. This time point was selected based on the replication kinetics of CA09wt virus in rhesus macaques. Virus was detected in all tissues and secretions from the mock-immunized group (Table 2). In animals that received 1 dose of vaccine, challenge virus was detected in the nasal turbinates of one animal, while two other animals showed low titers of virus in the nasal swabs and high titers of virus in the lungs. Two doses of vaccine elicited better protective efficacy; virus was only detected in nasal swabs and lung tissue from one animal; this animal had a lower serum HI antibody titer than the other animals (HAI titer =160) (Table 2), and no pH1N1 specific IgA antibodies detected in the nasal wash (Figure 4B (day 56))
In this study, we compared the pathogenesis of the 2009 pandemic H1N1 (CA09wt) virus in two species of non-human primates, African green monkeys and rhesus macaques. Non-human primates support replication of influenza viruses for a short duration but virus replication may be detected without evidence of clinical illness. The more recent studies have been conducted in Old World monkeys; cynomolgus macaques and Rhesus macaques have been used to study HPAI H5N1 and 1918 pH1N1 virus infection. These monkeys exhibited systemic symptoms similar to those experienced by humans, including fever, lethargy, and the loss of appetite, but respiratory signs and symptoms were limited [14, 24, 25]. Cynomolgus macaques infected with a seasonal H3N2 virus did not show clinical signs of infection, despite the recovery of virus from nasal swabs and lung lavage  whereas seasonal H1N1 virus infected pigtailed macaques showed clinical signs of illness, including weight loss, nasal discharge and fever associated with lung pathology . Thus, the demonstration of clinical illness in non-human primates is determined by the species, the route of inoculation and the influenza virus strain . In our study, none of the animals receiving the pH1N1 virus developed clinical symptoms although viral replication was observed in the respiratory tract, which was similar to what was observed in ferrets and mice infected with the CA09wt virus . However, our findings differ from the observations previously reported in cynomolgus macaques. Cynomolgus macaques infected with the pH1N1 virus showed clinical signs of disease progression [11–13]. The discrepancy between our findings and those in cynomolgus macaques may be because the susceptibility of different species of non-human primates differs or because different routes and dose of inoculum were used. In two of the cynomolgus macaque studies, virus was administered by multiple routes including intratracheal, intranasal, ocular or conjunctival and oral simultaneously, whereas in our study we inoculated via I.T. and I.N. routes only. Heterogeneity of viral replication in different non-human primate species raises the possibility that host factors such as the influenza virus receptor pattern differs among non-human primate species. The distribution of influenza virus receptors in different non-human primate species needs to be explored further.
Lung consolidation has been described as a feature of severe influenza virus infection in humans [28–31]. Macrophages and neutrophils are prominent cell types associated with and that potentially mediate severe lung pathology following infection with highly virulent H5N1 and 1918 influenza viruses in mice . Evidence of macrophage and neutrophil infiltration in the lungs was also demonstrated in cynomolgus macaques [14, 24] infected with highly pathogenic H5N1 and 1918 viruses. In the present study, we determined the cellular composition in the lungs of AGMs and rhesus macaques following pandemic H1N1 virus infection. The cellular composition in the lungs of CA09wt infected rhesus macaques (Fig. 3B) was similar to that observed in the lungs of mice infected with 1918 pH1N1 and highly pathogenic H5N1 viruses , where a dramatic increase of macrophages was observed in the lungs on day 7 post-infection. We did not observe macrophage infiltration in the lungs of CA09ca-infected rhesus macaques. We suspect that this is because the CA09ca virus is temperature sensitive and does not replicate in the lungs. Therefore, the infiltration of macrophages may be a consequence of the replication of wild type virus in lungs of AGMs and rhesus macaques. However, in the current study we did not determine the function of these cells in pH1N1 infection in AGMs and rhesus macaques.
We and others have demonstrated the efficacy and immunogenicity of the live attenuated CA09ca vaccine in small animal models such as mice and ferrets [5, 33]. In this study, we examined the efficacy and immunogenicity of the CA09ca vaccine in rhesus macaques because we found that these animals support CA09wt virus replication better than AGMs. Because we observed no clinical signs of disease in this model, we evaluated the efficacy of vaccine based on protection from replication of the CA09wt challenge virus. Although the CA09ca vaccine showed minimal replication limited to the upper respiratory tract of rhesus macaques, serum HAI as well as H1N1-specific mucosal IgA antibodies were detected in animals that received only one dose of vaccine and the titers of these antibodies increased after the second dose. These results indicate that the CA09ca vaccine is immunogenic in rhesus macaques. Animals receiving either one or two doses of vaccine were protected from CA09wt virus challenge. We also observed an inverse correlation between serum HAI and nasal IgA antibodies and the degree of protection from replication of the challenge virus, suggesting that the humoral immune response plays a critical role in protection.
In summary, our results indicate that the virus as well as non-human primate species determines the pattern of influenza virus infection and that the live attenuated CA09ca vaccine virus is immunogenic and efficacious in rhesus macaques.
This research was performed as part of a Cooperative Research and Development Agreement between the Laboratory of Infectious Diseases, NIAID and MedImmune. This work was supported in part by the Intramural Research Program of National Institute of Allergy and Infectious Diseases (NIAID), NIH. We would like to thank Jin Hong and George Kemble, MedImmune, Inc for critical reading of this manuscript.
We would like to thank Dr. Richard Herbert, Joanne Swerczek, Mark Szarowicz and staff of the NIH animal center at Poolesville for excellent technical support in the animal studies. We also thank Dr. Fernando Torres-Velez, Comparative Medicine Branch, NIH/NIAID/DIR and Dr. Ian Moore, LID/NIAID for technical assistance with necropsies.
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