Early viral exposure in mice weakens vaccine response, raising questions for human studies

In a preprint* research paper recently uploaded to the bioRxiv server, researchers at Washington University investigated how the exposome can mediate immune function in house mice, probably the most widely used model systems for in vivo immunological experimentation. Mice were sequentially inoculated with six different viral pathogens from formative years (neonatal) stages, following which their immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was measured. Their results suggest that viral exposure during formative years stages can substantially reduce antibody responses to vaccination, making prior pathogen exposure a vital consideration in murine model vaccine studies.

Study: Sequential early-life viral infections modulate the microbiota and adaptive immune responses to systemic and mucosal vaccination. Image Credit: Christoph Burgstedt / Shutterstock

*Essential notice: bioRxiv publishes preliminary scientific reports that usually are not peer-reviewed and, subsequently, mustn’t be considered conclusive, guide clinical practice/health-related behavior, or treated as established information.

The exposome

Murine models, especially those using house mice (Mus musculus), are among the mostly utilized in vivo systems for immunological and biomedical research. Mice have a brief generation time, are easy to rear and experiment upon under controlled laboratory environments, well-characterized phenotypically and physiologically represent human immunological responses. This makes these animals versatile and reliable model systems to check vaccines, pharmaceutical drugs, and immunological modalities.

Recent research has identified a possible oversight in the standard use of murine models – laboratory mice are reared in tightly controlled, specific pathogen-free (SPF) animal facilities, thereby stopping their exposure to natural microbes that humans and wild mice encounter under normal conditions. Despite genotypic and immune system structure and performance commonalities between laboratory mice and their wild counterparts, a growing body of evidence elucidates that pathogen exposure in the previous invokes different immunological responses than those observed within the latter.

The exposome is a comparatively novel concept, defined by Miller and Jones in 2014 because the “cumulative measure of environmental influences and associated biologic responses throughout the life span, including exogenous exposures and endogenous processes.” The exposome is thus the sum of all external and internal chemical, physical, biological, and social aspects influencing health. Since wild mice are exposed to a plethora of naturally occurring microbial influences that their laboratory-raised counterparts never encounter, the exposome concept hypothesizes that these cohorts would depict observable differences of their immune response to viral or pathogenic inoculation.

Scant research into this association has hitherto been unable to experimentally confirm this expectation, with studies being unable to determine differential immunological trends between wild- and laboratory-reared animals. These studies have focused on adult mice, which can fail to account for early-life changes that microbial exposure might modulate. There exists a necessity for studies to check how developmental exposure might ‘prime’ the immune systems of mice, potentially differing from observations from adult exposure where pathogenic priming may not alter immune function.

“In an effort to develop a tractable sequential infection model for broad laboratory use, in addition to to further describe the microbial and immune changes that result from sequential microbial exposures, we devised a virus-only sequential infection model starting in formative years that’s accomplished by 6 weeks of age, allowing mice for use for further experiments in a rapid and well-controlled manner.”

In regards to the study

The current study goals to plan a murine immune system priming model that’s initiated within the neonatal stage and persists across life. Sequential viral inoculation is anticipated to cause observable changes in immune cell population composition and antibody and cytokine expression levels. If successful, this might form the premise for a traceable ‘humanized’ model of immune response that higher reflects the real-life outcomes of vaccination.

Researchers used a case-control study design wherein wild-type (WT) C57BL/6J mice were allowed to mate. Their progeny were divided into the sequentially infected case-cohort and controls raised under conventional SPF methodology. Case mice were inoculated at age seven days using six viral pathogens – murine rotavirus strain (MRV), murine gamma-herpesvirus 68 (MHV68), murine norovirus strain CR6 (MNV), influenza virus strain PR8 (IAV), coxsackievirus B3 (CVB3), and murine astrovirus (MAstV). Inoculation was conducted sequentially at one-week intervals.

Fecal and blood samples were periodically collected for immunological measurements. Following final inoculum exposure, mice were allowed 4 weeks of recovery, after which ChAd-SARS-CoV-2-S viral particles were introduced via intramuscular injection to each cohorts to simulate pathogenic vaccination.

To research complete blood counts (CBC) and differential white blood cell (WBC) composition between test and control mice cohorts, hematological evaluation was used. Enzyme-linked immunoassay (ELISA) experiments were used to discover antibody specificity following vaccination. Flow cytometry was employed to discover and characterize splenocytes, tissue cells, and peripheral blood leukocytes. Multiplex immunoassays and mouse Antibody Isotyping Panel (AIP) were used to measure serum chemokines, antibodies, and cytokines. Intercellular cytokine staining and peptide restimulation assay were conducted to confirm these outcomes.

16S rRNA gene Illumina sequencing was carried out to discover and quantify viral DNA present in mouse fecal samples. Finally, statistical significance testing was employed to characterize and discuss differences in observed outcomes between case and control mice.

Study findings

“We sought to develop an “immunologically-mature” murine model in a genetically defined background by utilizing a well-controlled series of microbial exposures, focused exclusively on viruses, that might be rapidly administered to allow further experimental intervention by the age of 10 weeks.”

Sequential viral exposure was found to create a persistent pro-inflammatory host environment in case mice in comparison with their control counterparts. Global immunological changes were observed in case mice with hematological evaluation, revealing that at week 10, leukocytes were significantly unregulated compared to controls raised under aseptic conditions. While WBC proportions remained unchanged, their absolute numbers increased substantially in case mice. Serum cytokine evaluation revealed drastic increases in pro-inflammatory cytokines interleukin (IL)-6, interferon (IFN)-g, and tumor necrosis factor at nine weeks of exposure, which together reveal enhanced immune response in viral-exposed mice.

Sequential viral infection was found to manage the circulating and tissue-resident components of adaptive immunity in case mice. Viral exposure was moreover observed to modulate intestinal microbiome composition. In contrast, SPF mice were found to point out minimal variation in gut microbiome composition. Inoculation with the SARS-CoV-2 vaccine was found to limit antibody response and unregulate T-cell expression within the case-cohort, reducing symptomatic presentation and potentially reducing vaccine efficacy.

Conclusions

This preprint presents the primary evidence of the results of viral exposure across life stages, especially in neonatal and formative years periods. Researchers sequentially exposed one-week-old mice to 6 viral pathogens, following which intramuscular administration of a SARS-CoV-2 vaccine was undertaken. Unlike previous work, substantial differences between cytokines, WBCs, and other immune-modulating components were observed between sequentially inoculated mice and their SPF counterparts.

“Rodent models with heightened microbial exposures, either via cohousing with “dirty” mice or via sequential infection, have been shown to exhibit diminished humoral responses to vaccination.”

Study findings mirror these results and suggest that while innate and adaptive immunity is stronger in mice exposed to viral exposure in formative years, their response to vaccination is blunted, highlighting that in vivo vaccine testing in SPF murine models overestimates vaccine efficacy, a trend that likely extends to humans.

“Overall, the outcomes of this study indicate that sequential viral infections modulate the microbiota and result in changes within the immune system that dampen specific adaptive responses to systemic and mucosal vaccination. This study highlights the importance of early-life microbial exposure and its impact on the immune system and gut microbiota. Sequential infection provides a strong model for a matured immune system that will be readily leveraged for immunology, virology, and vaccine studies.”

*Essential notice: bioRxiv publishes preliminary scientific reports that usually are not peer-reviewed and, subsequently, mustn’t be considered conclusive, guide clinical practice/health-related behavior, or treated as established information.