Pandemic 1918 Influenza Virus Does Not Cause Lethal Infection in Rhesus or Cynomolgus Macaques

Abstract

The 1918 H1N1 influenza pandemic was among the most severe in history, taking the lives of approximately 50 million people worldwide, and novel prophylactic vaccines are urgently needed to prevent another pandemic. Given that macaques are physiologically relevant preclinical models of human immunology that have advanced the clinical treatment of infectious diseases, a lethal pandemic influenza challenge model would provide a stringent platform for testing new influenza vaccine concepts. To this end, we infected rhesus macaques and Mauritian cynomolgus macaques with highly pathogenic 1918 H1N1 influenza virus and assessed pathogenesis and disease severity. Despite infection with a high dose of 1918 influenza delivered via multiple routes, rhesus macaques demonstrated minimal signs of disease, with only intermittent viral shedding. Cynomolgus macaques infected via intrabronchial instillation demonstrated mild symptoms, with disease severity depending on the infection dose. Cynomolgus macaques infected with a high dose of 1918 influenza delivered via multiple routes experienced moderate disease characterized by consistent viral shedding, pulmonary infiltrates, and elevated inflammatory cytokine levels. However, 1918 influenza was uniformly nonlethal in these two species, demonstrating that this isolate is insufficiently pathogenic in rhesus and Mauritian cynomolgus macaques to support testing novel prophylactic influenza approaches where protection from severe disease combined with a lethal outcome is desired as a highly stringent indication of vaccine efficacy. IMPORTANCE The world remains at risk of an influenza pandemic, and the development of new therapeutic and preventative modalities is critically important for minimizing human death and suffering during the next influenza pandemic. Animal models are central to the development of new therapies and vaccine approaches. In particular, nonhuman primates like rhesus and cynomolgus macaques are highly relevant preclinical models given their physiological and immunological similarities to humans. Unfortunately, there remains a scarcity of macaque models of pandemic influenza with which to test novel antiviral modalities. Here, we demonstrate that even at the highest doses tested, 1918 influenza was not lethal in these two macaque species, suggesting that they are not ideal for the development and testing of novel pandemic influenza-specific vaccines and therapies. Therefore, other physiologically relevant nonhuman primate models of pandemic influenza are needed.

Keywords: 1918 influenza; cynomolgus macaques; influenza model; rhesus macaque.

FIG 1

Rhesus macaques infected with 1918 influenza virus. (A) Study overview of four rhesus macaques infected with 7 × 10⁶ PFU of 1918 influenza virus via intranasal, ocular, oral, and intratracheal routes. Blood, vitals, and swabs were collected on the indicated days postinfection. (B to G) Readouts of infection severity for weight loss (B), respiratory rate (C), rectal temperature (D), heart rate (E), oxygen saturation (SPO₂) (F), and clinical score (G). (H to J) Levels of influenza viral genomes were measured by RT-qPCR in nasal swabs (H), throat swabs (I), and rectal swabs (J). Only samples with values above the limit of detection of the assay (9.3 × 10² genome copies) are shown. bpm, beats per minute.

FIG 2

Experimental overview of 1918 influenza virus-infected cynomolgus macaques. Three groups of four cynomolgus macaques were infected with various doses of 1918 influenza virus via multiple routes of inoculation, including intrabronchial, ocular, intranasal, oral, and intratracheal. Sampling days are indicated, and samples collected included blood, bronchoalveolar lavage (BAL) fluid, X ray, and nasal, oral, and throat swabs.

FIG 3

1918 influenza virus infection of Mauritian cynomolgus macaques by multiple doses and routes of exposure. Group 1 animals (n = 4) were infected with 5 × 10⁴ PFU via intrabronchial inoculation, group 2 animals (n = 4) were infected with 5 × 10⁵ PFU via intrabronchial inoculation, and group 3 animals (n = 4) were infected with 7 × 10⁶ PFU via intranasal, ocular, oral, and intratracheal inoculation. Weight loss (A), heart rate (B), respiratory rate (C), oxygen saturation (SPO₂) (D), rectal temperature (E), and clinical score (F) were monitored for the duration of the study for all animals. Data were compared by two-way ANOVA with Tukey’s multiple-comparison test between the groups. Symbols for group 1 versus group 2 (#) and group 1 versus group 3 (*) represent P values of <0.05.

FIG 4

Viral detection in nasal and throat swabs from 1918 influenza virus-infected animals. Three groups of cynomolgus macaques (n = 4) were infected with either 5 × 10⁴ PFU (group 1) or 5 × 10⁵ PFU (group 2) of 1918 influenza virus via intrabronchial inoculation or 7 × 10⁶ PFU (group 3) of virus via intranasal, ocular, oral, and intratracheal inoculation. (A and B) Levels of viral RNA were determined by RT-qPCR in the nasal (A) and throat (B) swabs collected from each animal. Only samples with values above the limit of detection of the assay (9.3 × 10² genome copies) are shown. (C and D) Infectious virus in the PCR-positive nasal (C) and throat (D) swabs was measured by TCID₅₀ assays.

FIG 5

Thoracic radiograph scoring of Mauritian cynomolgus macaques infected with 1918 influenza virus. Three groups of cynomolgus macaques (n = 4) were infected with either 5 × 10⁴ PFU (group 1) or 5 × 10⁵ PFU (group 2) of 1918 influenza virus via intrabronchial inoculation or 7 × 10⁶ PFU (group 3) of virus via intranasal, ocular, oral, and intratracheal inoculation. (A) Thoracic radiographs taken on sampling days were scored for the presence of pulmonary infiltrates in each of the lung lobes, and scores were totaled to give a single score on each examination day that could range from 0 to 18. (B) Thoracic radiographs were taken of animals with peak radiographic scores on day 8 postinfection from groups 1, 2, and 3.

FIG 6

Cytokine expression in the bronchoalveolar lavage fluid samples of Mauritian cynomolgus macaques infected with 1918 influenza virus. Three groups of cynomolgus macaques (n = 4) were infected with either 5 × 10⁴ PFU (group 1) or 5 × 10⁵ PFU (group 2) of 1918 influenza virus via intrabronchial inoculation or 7 × 10⁶ PFU (group 3) of virus via intranasal, ocular, oral, and intratracheal inoculation. Individual cytokine expression levels were determined using a cytokine monkey magnetic 29-plex panel. Data were analyzed by two-way ANOVA with Tukey’s multiple-comparison test. Data are shown as means and SEM. *, #, and § represent significance within group 1, group 2, and group 3, respectively, when each time point was compared with day 0. One, two, and three symbols represent P values of <0.05, <0.01, and <0.001, respectively.