Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques

1Coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China and spread throughout the world causing a global pandemic. Some patients who were discharged with undetectable SARS-CoV-2 have reportedly had a positive result upon subsequent tests. Recently, SARS-CoV-2-specific neutralizing antibodies (NAbs) were detected around 10–15 days after the onset of COVID-19. The possibility that patients have a risk of “relapse” or “reinfection” after recovery from the initial infection has raised concern. In this study, we therefore used nonhuman primates to longitudinally track the short-term infectious status from primary SARS-CoV-2 infection to reinfection by the same viral strain.

2Seven adult Chinese-origin rhesus macaques (M0 to M6, 3–5 kg, 3–5 years of age) were modeled for challenge–rechallenge observations. Six monkeys (M1 to M6) were intratracheally challenged with SARS-CoV-2 at 1 × 106 50% tissue-culture infectious doses (TCID50). After undergoing a mild-to-moderate course of SARS-CoV-2 infection, and transitioning into the recovery stage from the primary infection, four monkeys (M3 to M6) were rechallenged intratracheally with the same dose of the SARS-CoV-2 strain at 28 days post-initial challenge (dpi). The remaining two monkeys (M1 and M2) with primary infection were not rechallenged and were used as the negative control of the rechallenge group. A healthy monkey (M0) was given an initial challenge as a model control of the second challenge. The pathological changes with viral-dependent distribution were compared using necropsy specimens between two monkeys that underwent only the initial challenge (M0 at 5 dpi and M1 at 7 dpi) and two monkeys that underwent challenge–rechallenge (M3 and M5) at 5 days post rechallenge (dpr, 33 dpi). Body weight, rectal temperature, nasal/throat/anal swabs, hematological measurement, chest X-ray, virus distribution, pathological changes, and the analysis of immunocytes, and binding and neutralizing antibodies were examined at the designated time points (Fig. 1). Weight loss ranging from 200 to 400 g was found in four monkeys that underwent the initial challenge (4/7, M0, M1, M2, and M4) (Fig. 2A), whereas the rectal temperature was not elevated in any of the monkeys (0/7) (Fig. 2B). Reduced appetite and/or increased respiration were common (6/7, the exception of M4), but emerged transiently and exhibited a very short duration. Regarding viral dissemination, the peak viral load (6.5 log10 RNA copies/mL) in nasal swabs and pharyngeal swabs was detected at 3 dpi, followed by a gradual decline (Fig. 2, C and D). The peak viral load (5 log10 RNA copies/mL) in anal swabs was observed at 3 dpi, followed by a linear decline to reach undetectable levels at 14 dpi (Fig. 2E). In all monkeys that received the initial challenge, white blood cell count (WBC, 3.5–9.5 × 109/L), lymphocyte counts (LYMP, 1.1–3.4 × 109/L), and neutrophil counts (NEUT, 1.8–6.4 × 109/L) fluctuated within normal ranges. Compared with the baseline, a slight but significant reduction in WBC and LYMP was observed after the primary infection (Fig. 2F). On radiological examination, bilateral obscured diaphragmatic surface and decreased transparency of lung fields with a small patch shadow in the left lower lobe were detected, indicating mild-to-moderate interstitial infiltration in monkeys with pneumonia (represented by M4 and M6, Fig. 2G). Using necropsy specimens, viral RNA copies were detected in the M0 monkey at 5 dpi and the M1 monkey at 7 dpi in the nose (106–108 copies/mL), pharynx (104–106 copies/mL), lung (103–107 copies/mL), and gut (104–106 copies/mL) (Fig. 3A, left panel). Hematoxylin and eosin (H&E) staining revealed a mild-to-moderate interstitial pneumonia characterized by widened alveolar septa, increased alveolar macrophages and lymphocytes in the alveolar interstitium, and degenerated alveolar epithelia; moreover, infiltrated inflammatory cells were detected in the lungs of monkeys with primary infection. Collagen fiber could also be observed in the thickened alveolar interstitium in M0 and M1 monkeys using Modified Masson’s Trichrome stain at 5 or 7 dpi (Fig. 3B). Furthermore, the mucous membranes of the trachea, tonsils, pulmonary lymph nodes, jejunum, and colon of the M0 and M1 monkeys exhibited inflammatory cell infiltrations (fig. S1, left panel), as well as infiltration with abundant CD4+ T cells, CD8+ T cells, B cells, macrophages, and plasma cells in lungs, as assessed using immunohistochemistry (IHC) (fig. S2). Viral-infected cells were mainly found in alveolar epithelia and macrophages by IHC on sequential sections (Fig. 3C), as well as in the mucous membranes of the trachea, tonsils, pulmonary lymph nodes, jejunum, and colon (fig. S1), confirming that SARS-CoV-2 caused COVID-19 in rhesus monkeys. Collectively, these data demonstrated that all seven monkeys were successfully infected with SARS-CoV-2 and that the pathogenicity in monkeys is similar to that reported in recent studies.

3By 15 dpi, the body weight of infected monkeys (M2 to M6) had gradually increased into the normal range (4/5, the exception of M4, Fig. 2A), and their rectal temperature was maintained within the normal range (Fig. 2B). Moreover, the viral loads were negative in all nasopharyngeal and anal swabs (5/5, Fig. 2, C to E). As shown in Fig. 2F, the hematological changes remained relatively stable within the normal range. Chest X-rays returned to normal levels at 28 dpi (represented by M4 and M6, Fig. 2G). These traits were similar to the hospital discharge criteria used for patients with COVID-19, including absence of clinical symptoms, radiological abnormalities and twice-negative RT–PCR results. Taken together, our results suggest that monkeys that underwent initial SARS-CoV-2 infection required about 2 weeks to transition into the recovery stage.

4At 28 dpi, four monkeys (M3 to M6) that underwent primary infection and recovery were rechallenged intratracheally with the same dose of an identical SARS-CoV-2 strain. The clinical tracking of the reinfection included examination of weight loss (Fig. 2A) and rectal temperature (Fig. 2B). Interestingly, the rechallenged monkeys exhibited a transient increase in temperature, which was not observed during the primary infection. Viral loads remained negative over a 2-week intensive detection of the virus in nasopharyngeal and anal swabs after rechallenge with SARS-CoV-2 (Fig. 2, C to E). Peripheral blood measurements revealed no significant fluctuation during the rechallenge stage (Fig. 2F). Moreover, we did not detect abnormalities by X-ray in the M4 and M6 monkeys at 33 dpi (5 dpr, Fig. 2G). In necropsy specimens of the lungs and extrapulmonary tissues of rechallenged monkeys (M3 and M5 at 5 dpr), we found no detectable viral RNA (Fig. 3A, right panel), no significant pathological lesions (Fig. 3B, right panel of fig. S1), no viral-infected cells (Fig. 3C, right panel of fig. S1), and no immune cell infiltration (fig. S2). Therefore, the rhesus monkeys that initially developed primary SARS-CoV-2 infection did not appear re-infected with the identical SARS-CoV-2 strain during their early recovery stage.

5To interpret the challenge–rechallenge disparity, we performed a comparison of the clinical, pathological, viral and immunological traits that comprehensively reflected the virus–host interaction between the primary-challenge stage and the rechallenge stage in four monkeys (M3 to M6). First, the viral loads in nasopharyngeal and anal swabs were much higher at 5 or 7 dpi than they were at 5 or 7 dpr (right side of Fig. 2, C to E). An increased WBC and neutrophils were observed at 14 dpr compared with 14 dpi (Fig. 2F, right side). Second, T and B cells from peripheral blood, including CD4+ T subsets [naïve CD4+ T cells (CD4+ T naïve), central memory CD4+ T cells (CD4+ Tcm), and effective memory CD4+ T cells (CD4+ Tem)], CD8+ T subsets (CD8+ T naïve, CD8+ Tcm, and CD8+ Tem), memory B cells, and plasma cells were relatively stable during the challenge–rechallenge infectious stage. However, an increased percentage of activated CD8+ T cells from peripheral blood was observed at 14 dpi, which was also found at 0 dpr compared with 0 dpi (Fig. 4A, right side). Regarding the immune responses from lymph nodes, an increased percentage of CD4+ Tcm cells and decreased percentage of naive CD4+ T cells and memory B cells from lymph nodes were observed at 5 dpr compared with 5 dpi (Fig. 4B). Third, the specific antibody levels against the SARS-CoV-2 spike increased gradually, leading to a significantly higher titer at 21 dpi than at 3 dpi and 42 dpi (14 dpr) compared with 28 dpi or 0 dpr (Fig. 4C). Moreover, the specific antibody titers were much higher at 14 dpr compared to 14 dpi (Fig. 4C, right panel). As shown in Fig. 4D, the average titers of neutralizing antibodies exhibited a linear enhancement at the time of rechallenge (28 dpi; range, 1:8-1:20 in all monkeys). We observed an enhanced activation of CD8+ T cells from peripheral blood, and changes in CD4+ Tcm cells and memory B cells from lymph nodes. It appeared an increased number of neutralizing antibodies against SARS-CoV-2 were induced by cellular or humoral immunity facilitated by the primary infection, which might have protected the same nonhuman primates against reinfection in the short term. However, factors that are directly correlated to protection are yet to be fully elucidated. Further studies of passive transfer of convalescent sera from this model to a naïve macaque, or CD8+ T cell depletion in the recovered monkeys prior to rechallenge, would be required to define the mechanisms underlying the pathogenicity of SARS-CoV-2.

6In the present challenge–rechallenge infection of rhesus monkeys with SARS-CoV-2, observations and detections were carried out within the relative short time window in which neutralizing antibodies plateaued after the primary infection. A longer interval (longer than 6 months) between the primary challenge and the rechallenge is needed to track longitudinally the host–virus interaction and elucidate the protective mechanism against SARS-CoV-2 in primates. Moreover, all infected monkeys exhibited relative mild-to-moderate pneumonia, which is similar to the mild or common clinical characteristics of COVID-19 in infected human individuals; however, a COVID-19 monkey or transgenic mouse model with severe clinical symptoms or lethality should be explored according to increasing challenge dose, exposed tissues, or other treatments. Although we observed macrophages in association with SARS-CoV-2 infection, rapid rechallenge in the absence of potent serology relieved some level of concern regarding the occurrence of ADE. In addition, mucosal immunity, which is triggered by primary infection and includes both the respiratory and intestinal mucosa and local lymph nodes, might contribute substantially to the viral rechallenge response. Thus, future studies are required to examine experimentally the mucosal antibody responses, such as bronchoalveolar lavage IgG levels, serum IgA or IgM levels.

7Taken together, our results suggest that rhesus macaques that have undergone an initial infection with SARS-CoV-2 mount protection against rechallenge during the early recovery days. However, it remains necessary to elucidate the protective mechanism against SARS-CoV-2 regarding neutralizing antibodies or other immunological roles. This short-term infection rechallenge macaque model provides insightful information for vaccination research, therapy of convalescent sera, and prognosis of COVID-19.