Refining the Cynomolgus Macaque Model for HPAI H5N1 Infection and Assessing the Efficacy of Seasonal Influenza Vaccination


The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the importance of developing effective vaccines and countermeasures against emerging viruses and bacteria that can rapidly spread within the human population.

Over the past quarter-century, there has been a growing concern about avian influenza viruses crossing the species barrier and infecting humans, particularly among poultry workers. While most cases of avian influenza in humans result in mild and self-limiting illness, highly pathogenic avian influenza (HPAI) H5N1 viruses have raised alarm due to their potential to cause severe respiratory disease with a high fatality rate.

This concern has led to efforts to better understand these viruses and develop vaccines and antiviral drugs to protect against them.

Influenza viruses have a remarkable ability to adapt to new hosts and reassort with one another, creating novel strains for which there is limited or no pre-existing immunity in the human population. Consequently, there is a significant fear that highly lethal avian influenza viruses could either adapt to mammalian hosts or reassort with human seasonal influenza viruses, potentially triggering a pandemic on the scale of the devastating 1918 influenza pandemic, which claimed the lives of millions worldwide. In this context, the urgent need for effective vaccines and antiviral therapies to combat avian influenza cannot be overstated.

Epidemiological studies have hinted at the possibility that early-life exposure to human influenza viruses may provide a form of immunological imprinting, potentially offering some degree of protection against closely related avian influenza viruses later in life. Likewise, research on ferrets has shown that prior exposure to H1N1 influenza can confer some protection against H5N1 challenge. However, whether vaccination against seasonal human influenza viruses could similarly protect against the lethality of avian influenza viruses remains uncertain.

Cynomolgus macaques have been utilized as a valuable model for studying severe influenza since the 1997 H5N1 outbreak in Hong Kong. These studies have revealed that cynomolgus macaques, when inoculated with high doses of H5N1 virus via mucosal routes such as intranasal, intratracheal, oral, and ocular, generally develop mild disease, with a relatively low fatality rate.

Importantly, avian influenza viruses, including HPAI viruses, have a predilection for binding to α2,3 sialic acids found predominantly in the lower respiratory tract of humans, and this pattern of sialic acid expression mirrors that in macaques.

To mimic a natural respiratory infection more accurately, previous studies have examined the inhalation of small-particle aerosols containing HPAI H5N1 virus. This method has the advantage of delivering the virus deep into the lung and distributing it more evenly throughout the organ.

Notably, earlier investigations found that inhalation of aerosolized HPAI H5N1 virus was highly lethal in cynomolgus macaques, with death occurring rapidly. There remained a critical question concerning whether the abbreviated disease course and severe outcome observed after aerosolized H5N1 infection were a consequence of the high doses used, and whether employing lower doses could extend the disease course and alter the outcome.

Lower doses could potentially be more useful in assessing vaccines or therapeutics, as they would better reflect natural exposure scenarios.

In this report, we aim to refine the cynomolgus macaque model of lethal H5N1 infection by investigating the relationship between inhaled dose and disease progression.

Additionally, we seek to assess whether vaccination against seasonal influenza using an adjuvanted quadrivalent influenza vaccine (aQIV) can provide protection against mortality and morbidity caused by HPAI H5N1 infection in macaques.


To refine the cynomolgus macaque model, we conducted a series of experiments using different doses of aerosolized HPAI H5N1 virus. The macaques were exposed to small-particle aerosols with varying viral loads to determine how the inhaled dose affected disease progression, including the severity and duration of symptoms.

Furthermore, we introduced a vaccination component to our study. We administered an adjuvanted quadrivalent influenza vaccine (aQIV) to a group of macaques and assessed whether this vaccination could confer protection against HPAI H5N1 infection. The vaccine contained antigens against seasonal influenza strains, and we aimed to investigate if it could induce cross-reactive immunity capable of mitigating the severity and lethality of H5N1 infection.


Our investigations into the relationship between inhaled dose and disease progression in cynomolgus macaques revealed several key findings. First, we observed that lower doses of aerosolized HPAI H5N1 virus led to an extended disease course compared to higher doses, suggesting that the severity of infection was dose-dependent. This observation has important implications for future studies evaluating potential vaccines and therapeutics, as it underscores the importance of using dose levels that more closely resemble natural exposure scenarios.

In the vaccination component of our study, we found that the administration of aQIV provided a degree of protection against HPAI H5N1 infection in macaques. While not all vaccinated macaques were completely immune to the virus, those that received the vaccine exhibited milder symptoms and a lower fatality rate compared to the unvaccinated control group. This suggests that vaccination against seasonal influenza may offer some cross-reactive immunity that can be beneficial in the context of avian influenza infection.


Sporadic yet recurring outbreaks of highly pathogenic avian influenza H5N1 (HPAI H5N1) in humans and its frequent spillovers to mammalian species have raised serious concerns about the potential for a future influenza pandemic. In the context of global public health, efforts to develop effective vaccine candidates that can provide broad protection against future pandemic influenza viruses are paramount.

Simultaneously, the development of model systems capable of evaluating these vaccine and drug candidates is essential, given that the efficacy of such countermeasures against pandemic influenza cannot be evaluated through human clinical trials alone.

This study aimed to validate the use of aerosol challenge with HPAI H5N1 virus in the cynomolgus macaque model as a surrogate for human pandemic influenza virus infection, particularly those associated with high morbidity and mortality. Furthermore, we explored whether repeated vaccination with an adjuvanted quadrivalent influenza vaccine (aQIV) could offer protection against mortality caused by H5N1 virus.

Our previous research had established that in cynomolgus macaques, exposure to high doses of aerosolized H5N1 resulted in fulminant, lethal viral pneumonia. This infection triggered a cytokine storm, inflammasome activation, pyroptosis of alveolar epithelial cells, and disruption of the epithelial barrier.

For this study, we aimed to investigate whether lower viral doses would extend the disease course and alter the outcome. Interestingly, even at the lowest doses tested, evidence of infection and viral replication was observed, albeit with only mild disease manifestations. We were able to establish an estimated lethal dose (LD50) for aerosolized H5N1, and macaques infected around this dose developed severe respiratory disease, with two-thirds succumbing to the disease within 4–6 days.

Lowering the viral dose did not substantially extend the disease course but resulted in comparatively milder disease within the same timeframe. It is noteworthy that recent studies by other groups exposed macaques and marmosets to H5N1 via aerosol, but their findings did not lead to lethal disease.

This discrepancy in results may be attributed to differences in aerosol generation methods, delivery mechanisms, and an unknown inhaled dose. The data presented here suggest that the clinical signs reported in those studies may be consistent with an inhaled dose of <5 log10 pfu.

One intriguing aspect of our study is the potential cross-reactivity of immunity generated by the aQIV vaccination. Despite the significant structural differences between the hemagglutinin (HA) of HPAI H5N1 and seasonal human H1N1 viruses, both H5 and H1 subtypes belong to group 1 HAs and share conserved epitopes, particularly in the stem region.

Prior research had shown that current seasonal influenza vaccines did not elicit protective immunity against lethal H5N1 challenge in animal models, including mice and ferrets. This was partly due to the inability of these animals to efficiently respond to the conserved HA stem epitope present in group 1 HA subtypes. However, macaques are capable of generating neutralizing antibodies targeting the HA stem epitope, which can provide prophylactic protection against H5N1 in other animal models.

In our study, we observed that aQIV-vaccinated macaques were indeed protected from mortality caused by aerosolized H5N1 infection. The levels of H5N1 virus neutralizing antibodies correlated with a reduction in both fever and virus titer in bronchoalveolar lavage samples. Interestingly, given the relatively weak neutralizing and HA-binding antibody titers to H5N1, it is possible that T cell responses played a significant role in the observed protection.

This scenario is reminiscent of how ancestral SARS-CoV-2 vaccines continue to prevent hospitalization and death even in the face of new variants poorly recognized by anti-spike antibodies. Alternatively, immune responses to other non-HA antigens, such as neuraminidase, may also contribute to the protection against mortality.

In conclusion, our refined semi-lethal aerosolized H5N1 macaque infection model holds great promise for studying the pathogenesis of this critical virus and for evaluating candidate vaccines and therapeutics. In the wake of the coronavirus pandemic, as well as a renewed emphasis on pandemic preparedness, extending the scope of this macaque model to explore the virulence of other influenza subtypes and strains, as well as the efficacy of medical countermeasures, should be a high priority. This research not only advances our understanding of avian influenza but also provides valuable insights and tools to address the ongoing threat of emerging infectious diseases.


The ongoing threat of avian influenza viruses crossing the species barrier and potentially triggering a devastating pandemic necessitates continuous research and preparedness efforts. Our study has refined the cynomolgus macaque model for HPAI H5N1 infection, providing a more accurate representation of natural exposure scenarios.

Additionally, it has demonstrated that vaccination against seasonal influenza using aQIV can confer some degree of protection against HPAI H5N1 infection, highlighting the potential utility of existing vaccines in mitigating the impact of avian influenza outbreaks.

As we continue to confront the ever-present risk of emerging infectious diseases, the knowledge gained from studies like this one will be essential in our efforts to develop effective countermeasures and protect global public health.

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