Sequential infection with H1N1 and SARS-CoV-2 aggravated COVID-19 pathogenesis in a mammalian model, and co-vaccination as an effective method of prevention of COVID-19 and influenza

Abstract

Influenza A virus may circulate simultaneously with the SARS-CoV-2 virus, leading to more serious respiratory diseases during this winter. However, the influence of these viruses on disease outcome when both influenza A and SARS-CoV-2 are present in the host remains unclear. Using a mammalian model, sequential infection was performed in ferrets and in K18-hACE2 mice, with SARS-CoV-2 infection following H1N1. We found that co-infection with H1N1 and SARS-CoV-2 extended the duration of clinical manifestation of COVID-19, and enhanced pulmonary damage, but reduced viral shedding of throat swabs and viral loads in the lungs of ferrets. Moreover, mortality was increased in sequentially infected mice compared with single-infection mice. Compared with single-vaccine inoculation, co-inoculation of PiCoVacc (a SARS-CoV-2 vaccine) and the flu vaccine showed no significant differences in neutralizing antibody titers or virus-specific immune responses. Combined immunization effectively protected K18-hACE2 mice against both H1N1 and SARS-CoV-2 infection. Our findings indicated the development of systematic models of co-infection of H1N1 and SARS-CoV-2, which together notably enhanced pneumonia in ferrets and mice, as well as demonstrated that simultaneous vaccination against H1N1 and SARS-CoV-2 may be an effective prevention strategy for the coming winter.

Fig. 1

Clinical findings of co-infection with H1N1 and SARS-CoV-2 in ferrets. a Experimental design and sample collection. Eighteen ferrets were used in this study. Six ferrets were inoculated intranasally with H1N1 (1 × 10⁶ TCID₅₀) and rechallenged intranasally with the same dose of SARS-CoV-2 (F/FC group, n = 6). The remaining ferrets were intranasally challenged with H1N1 (F/F group, n = 6) or SARS-CoV-2 (F/C group, n = 6) as control groups. Infected ferrets were observed for changes in body weight (b), body temperature (c), and clinical symptoms (d) at the indicated time points. Significant differences are indicated with asterisks (*P < 0.05, **P < 0.01; Student’s t test)

Fig. 2

Viral characteristics following co-infection with H1N1 and SARS-CoV-2 in ferrets. a Viral RNA loads of H1N1 from throat swabs were detected by qRT–PCR (n = 6). b Viral RNA loads of SARS-CoV-2 from throat swabs were detected by qRT–PCR (n = 6). c Viral RNA of SARS-CoV-2 was measured in individual lung lobes of infected ferrets (n = 4). Significant differences are indicated with asterisks (*P < 0.05, **P < 0.01; Student’s t test)

Fig. 3

Histopathological features of co-infection with H1N1 and SARS-CoV-2 in ferrets. a Histopathological changes of F/F, F/C, and F/FC ferrets at 5 days post-virus challenge. b Immunofluorescence analysis of H1N1 or SARS-CoV-2 antigens in lung tissues from the F/F, F/C, and F/FC ferrets. Black bar = 100 μm, white bar = 50 μm

Fig. 4

Co-infection of H1N1 and SARS-CoV-2 in K18-hACE2 mice. a Experimental design and sample collection in K18-hACE2 mice. b Changes in body weight and percent survival were recorded at the indicated time points in H1N1-infected K18-hACE2 mice (K18/F), SARS-CoV-2-infected K18-hACE2 mice (K18/C), and co-infected K18-hACE2 mice (K18/FC) (n = 6). c Pathogenesis of H1N1 and SARS-CoV-2 in K18/F, K18/C, and K18/FC mice. d Histopathological changes in K18/F, K18/C, and K18/FC dying mice. e Immunofluorescence analysis of K18/F, K18/C, and K18/FC mice. Black bar = 100 μm, white bar = 25 μm, yellow bar = 75 μm

Fig. 5

Comparison of cellular and humoral immunity among flu-vaccinated, SARS-CoV-2-vaccinated, or simultaneously immunized K18-hACE2 mice. a Flu vaccine immunized mice (K18/FV), PiCoVacc (whole SARS-CoV-2-inactivated vaccine) immunized mice (K18/CV), and simultaneously immunized mice (K18/FCV). H1N1-infected K18-hACE2 mice (K18/FM) and SARS-CoV-2-infected K18-hACE2 mice (K18/CM) were used as infected controls. b Neutralizing antibody titers were measured in immunized K18-hACE2 mice (n = 3). c Percentages of memory CD4⁺/CD8⁺ T-cell subsets from peripheral blood in co-infected mice (n = 4). d Changes in body weight and percent survival of mice immunized against SARS-CoV-2 or H1N1 infection at the indicated time points (n = 6, left panel). The viral RNA loads of H1N1 or SARS-CoV-2 were quantified at 5 dpi (n = 6, middle panel). The titers of neutralizing antibodies in K18-hACE2 mice (with or without vaccine immunization) before and after H1N1 or SARS-CoV-2 infection (n = 6, right panel). Significant differences are indicated with asterisks (*P < 0.05; **P < 0.01; Student’s t test)