dcyphr | SARS-CoV-2 infection protects against rechallenge in rhesus macaques


It is necessary to understand the protective immunity (how antibodies created against a primary infection respond to secondary infection) of SARS-CoV-2 to develop vaccine and health strategies to end the pandemic. An important question that has not been answered is whether SARS-CoV-2 infection results in protective immunity during a secondary infection. In this study, the researchers developed a rhesus macaque (a kind of monkey) model of SARS-CoV-2 infection. They found that the monkeys had a large amount of virus in the respiratory tract, humoral and cell-mediated immune responses, and evidence of pneumonia. After the virus was cleared from the monkeys’ systems, the researchers infected them again. They found that the amount of virus present in the respiratory system and mucus lining of the nose decreased. The immune response against secondary infection was mediated by the adaptive immune response. The results show that SARS-CoV-2 induces protective immunity during secondary infection in monkeys.


The researchers wanted to determine if SARS-CoV-2 infection would provide protective immunity against a secondary SARS-CoV-2 infection in rhesus macaques.


The rapid spread of COVID-19 has made the development of vaccine and health strategies a global priority. However, understanding of immune responses against SARS-CoV-2 is limited. It is unknown whether primary SARS-CoV-2 infection provides protective immunity against re-exposure in humans. This information is important for vaccine development, modeling of disease transmission, and public health strategies. In this study, the researchers develop a rhesus macaque model of SARS-CoV-2 infection and try to understand various features of infection and assess whether protective immunity is created or not.


Virology and immunology of SARS-CoV-2 infection in rhesus macaques

After primary infection, the monkeys showed high levels of viral RNA in the lower respiratory system and in the mucus lining of the nose. Viral RNA increased over time, suggesting replication of the virus. The monkeys showed decreased appetite and responsiveness, suggesting a mild form of the disease. Extreme symptoms such as death and respiratory distress were not observed.

All monkeys developed antibodies against SARS-CoV-2 structural proteins and neutralizing antibodies, indicating activity in the humoral immune response. All monkeys also developed a cell-mediated immune response.

SARS-CoV-2 infection induces acute viral interstitial pneumonia in rhesus macaques

The researchers also assessed the pathologic (related to the cause and effects of diseases) characteristics of SARS-CoV-2 infection. They autopsied multiple regions of the body, and found high levels of viral RNA in the mucus lining of the nose, pharynx, trachea, and lung tissues. They found lower levels of viral RNA in the gastrointestinal tract, liver, and kidney.

The researchers found that multiple regions of the body showed evidence of inflammation and viral pneumonia. Virus-infected cells were found in the lungs, randomly dispersed, and were suggested to replicate. They were also associated with inflammatory particles in the lungs.

These results suggest that SARS-CoV-2 induced acute inflammation and viral pneumonia in many areas of the body that involved multiple cell types.

Protective efficacy against rechallenge with SARS-CoV-2

Upon secondary infection, the researchers observed very limited viral RNA on day 1 of reinfection and no viral RNA after. In controls, high levels of viral RNA was observed. Viral RNA was higher in the mucus lining of the nose than in the respiratory tract, but the researchers suggested that this was because the monkeys were infected through the nose. Little clinical disease was observed in the monkeys after reinfection.

After reinfection, the monkeys showed fast response by the adaptive immune system. All monkeys developed adaptive antibody responses following reinfection. The researchers suggest that the protective immunity against reinfection was mediated by rapid response of the adaptive immune system.


Some viruses do not generate protective immunity for reinfection, such as HIV. There is no data about whether people who have recovered from COVID-19 have protective immunity against SARS-CoV-2. In this study, the researchers demonstrate that rhesus macaques generate protective immunity against SARS-CoV-2 and are protected from reinfection.

The researchers developed a monkey model of SARS-CoV-2 infection that mimics human infection by the virus, including high levels of viruses in the respiratory tract and viral pneumonia. This suggests the potential of rhesus macaques as model organisms for immunological research and testing vaccines and therapeutics. However, no monkey models have led to respiratory failure or mortality. More research is necessary to create a monkey model of severe COVID-19 disease.

SARS-CoV-2 infection led to humoral and cell-mediated immune responses that provided protection against reinfection. However, because of the near-complete protection observed in the monkeys, the researchers were unable to determine the immune correlates (measurable signs that the monkeys are immune) of protection. The importance of neutralizing antibodies, other antibodies, cellular immunity, and innate immunity in protection must be determined. Additional research is necessary to understand the longevity of adaptive immune responses against SARS-CoV-2.

The results of this study suggest that immunological approaches to the prevention and treatment of COVID-19 is possible. However, because monkey models are so different from humans, clinical studies are necessary to determine whether SARS-CoV-2 infection provides protection against reinfection in humans.


Evaluating immune response against primary infection

The researchers infected 9 monkeys with various amounts of the SARS-CoV-2 virus through the nose and trachea. After infection, they quantified the virus by assessing viral RNA levels using PCR.

To distinguish viruses used for infection and replicated virus, the researchers assessed E gene subgenomic mRNA (sgmRNA) using PCR. E gene subgenomic mRNA is only found in replicated viruses.

The researchers evaluated the humoral immune response in infected monkeys using ELISA, a pseudovirus neutralization assay, and a live virus neutralization assay. They evaluated the cell-mediated immune response using intracellular cytokine staining assays.

Evaluating pathologic characteristics

The researchers infected 4 monkeys with SARS-CoV-2 and autopsied them on day 2 of infection.

The researchers fixed tissue from the mucus lining of the upper airway, the trachea, and the lungs with formaldehyde and “froze” them in paraffin to understand the inflammatory response and diagnose pneumonia.

Immunohistochemistry and RNAscope were used to detect clusters of virus-infected cells in the lungs.

Cyclic immunofluorescence (CyCIF) was used to further characterize infected tissues.

Evaluating immune response against secondary infection

35 days after primary infection, the researchers re-infected the monkeys. They also infected 3 monkeys who did not undergo primary infection to serve as controls.