Immunology taught by vaccines

Despite the success of vaccination in controlling many infectious diseases, there are challenges in designing vaccines against HIV, malaria and tuberculosis, and other pathogens that, in aggregate, afflict billions of people. Vaccine development has been frustrated by a lack of detailed understanding about what types of immune responses are effective at preventing infection, and failure to translate successes in animal models to humans. This problem is compounded by the variability in vaccine efficacy in humans. For example, vaccines against oral pathogens such as Rotavirus and Poliomyelitis have considerably lower efficacy in children in some low- and middle-income countries (LMICs) compared to those in high-income countries (1). Environmental differences, persistent parasitic infections, malnutrition, and environmental enteropathy may affect immune system function and its ability to respond to vaccination (1, 2). Several studies have also suggested that intestinal microbiota composition plays a role (1, 2).

The microbiota consists of trillions of bacteria in the human gut and in other peripheral tissues such as the skin and lungs, and they outnumber human cells 10-fold (3). Emerging evidence suggests a potent role for this microbial universe in shaping many aspects of physiology, including the immune system, the cardiovascular system, and various aspects of host metabolism (2, 3). Loss of microbial diversity in microbiota (dysbiosis) is strongly associated with many inflammatory diseases, and intestinal dysbiosis is linked to reduced efficacy of various immune interventions, including prevention of HIV infection and immune checkpoint blockade, a form of cancer immunotherapy. However, much of the evidence for this comes from elegant studies in mice administered antibiotics to deplete their microbiota, or in germ-free mice that are devoid of any bacteria from birth. The extent to which these results reflect what happens in humans is uncertain. Many studies have established correlations between particular species of bacteria and various aspects of human physiology, but there remains little causal evidence.

Immunologists have recently harnessed vaccines as probes to study the human immune system. Systems biological approaches that can measure the expression of genes, proteins, metabolites, and immune cell types have been used to study immune responses to vaccination. The first examples of such studies used the live attenuated yellow fever vaccine (YF-17D) (4, 5), which has been administered to 600 million people worldwide and is one of the most effective vaccines developed. Blood gene expression “signatures” induced within a few days of vaccination with YF-17D correlated with the ensuing CD8⁺ T cell and neutralizing antibody response. Machine learning techniques delineated signatures capable of predicting the immunogenicity of the vaccine in individuals (4). Subsequently, systems approaches have been used to identify signatures that predicted the immunogenicity of other vaccines, such as the inactivated seasonal influenza vaccine or malaria vaccine, in diverse populations such as the elderly, infants, and identical twins and in geographically distinct populations (6–8). Such signatures reflect a range of biological processes, such as the early innate immune response and amino acid starvation response, as well as signatures of the antibody-producing cell response (4–8).

Several mechanistic insights have emerged from these studies. One such insight concerns the impact of the microbiota on vaccination in humans. Analysis of blood transcriptional responses induced by vaccination of healthy adults with the inactivated seasonal influenza vaccine revealed a strong correlation between the early expression of the gene encoding Toll-like receptor 5 (Tlr5), an innate immune receptor that senses bacterial flagellin, on day 3 after vaccination and the ensuing vaccine-specific antibody titers measured 28 days after vaccination (9). The association between the antibody response to a viral vaccine and the expression of a receptor involved in bacterial sensing was puzzling and suggested a potential link between microbiota and vaccination-induced immunity. Vaccination of mice genetically deficient in Tlr5 (Tlr5^−/− mice) resulted in impaired antibody responses (9). Antibody responses to influenza vaccination were also impaired in germ-free mice and mice administered broad-spectrum antibiotics. Moreover, Tlr5^−/− mice and antibiotic-administered mice had impaired antibody responses to the inactivated polio vaccine, which, like the inactivated seasonal influenza vaccine, contains no exogenous adjuvants (or immuneboosting agents). By contrast, Tlr5^−/− mice or antibiotic-administered mice were capable of mounting normal antibody responses to vaccines that contained adjuvants such as the alum-adjuvanted tetanus, diphtheria, and pertussis vaccines (9). This raised the concept that the microbiota could be serving as “endogenous adjuvants” that boost immunity to vaccines lacking adjuvants.

The possibility that the microbiota plays a role in modulating immune responses to vaccination in humans is of particular concern because vaccines are less effective in many regions of LMICs, where widespread and indiscriminate use of antibiotics, particularly in neonates and infants, could cause long-lasting changes in the microbiota (1). However, the effects of the antibiotic azithromycin were examined on the immunogenicity of oral polio vaccine in seronegative infants in India, and the antibody response was unaffected (10). Of note, detailed analysis of immune responses was not performed in this study, so further investigation is warranted.

To comprehensively assess the impact of the microbiota in humans, a clinical trial was performed in which a cocktail of broad-spectrum antibiotics was given to healthy young adults before and after vaccination with the inactivated seasonal influenza vaccine (11). Antibiotics administration resulted in a 10,000-fold reduction in the total quantity of gut bacteria, but this effect was shortlived and normal numbers were regained within a few days. Furthermore, gut bacterial diversity was reduced, and this took longer than 6 months to recover (11). Nonetheless, the antibody responses to vaccination were unaffected. The individuals in this study had high concentrations of influenzaspecific antibodies prior to vaccination, indicating recent exposure to the vaccine or influenza virus. Another clinical trial was conducted in which anyone who had either received the influenza vaccine or had been exposed to influenza during the past 3 years was excluded. There was a marked impairment in the immunoglobulin G1 (IgG1) and IgA antibody response specific to the H1N1 strain of influenza, as well as in antibody neutralization to the H1N1 strain (11). In humans, the serum antibody response to vaccination with seasonal influenza vaccine is dominated by the IgG1 subclass (with lower contributions from IgM and IgA antibodies), and IgG1 antibodies can neutralize the virus as well as mediate a range of effector cell functions. Curiously, the impairment in IgG1 and IgA responses was only observed against one of the three influenza strains contained in the vaccine—the H1N1 strain—and not against the H3N2 or B strains. The reason for this is unclear, but it is possible that adults have high H3N2 and B subtype immunological memory due to prior exposure, and thus a higher threshold of memory against these strains could withstand the effects of antibiotics. Thus, the adaptive immune response truly does seem to be adaptive, and resilient to even the most severe perturbations in the microbiota. This resilience of the adaptive immune system is evident in the context of genetic variation. Thus, a previous study has demonstrated that responses to influenza vaccination in identical twins were largely driven by environmental factors such as prior immune exposure and were independent of genetic factors (8).

In addition to these effects on the adaptive immune response, analysis of transcriptional signatures revealed that antibiotics treatment resulted in enhanced innate immune responses, specifically inflammation and in particular several gene expression programs associated with the transcription factors activating protein 1 (AP-1, comprising FOS and JUN) and nuclear receptor 4A1 (NR4A1), which play central roles in mediating inflammatory responses (11) (see the figure). These same transcriptional modules were increased in healthy elderly subjects immunized with the seasonal influenza vaccine (6). These results indicate that antibiotics-driven depletion of the gut microbiota may drive inflammatory responses to vaccines similar to age-associated increases in inflammation and that frequent and longterm usage of antibiotics may accelerate the process called “inflammaging,” a chronic low-grade inflammation that can develop with advanced age and contribute to the pathogenesis of age-associated diseases (12). Future studies should thus explore the potential connection between antibiotics usage, inflammaging, and impaired vaccine immunity in the elderly.

Figure. The gut microbiota affects vaccination.

In healthy humans, the gut microbiota enhances antigen-specific immunoglobulin G1 (IgG1) and IgA antibody responses to vaccination. Secondary bile acids metabolized by gut microbiota suppress excessive inflammation driven by NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome and activating protein 1 (AP-1)-associated gene expression.

In addition to the effects on the adaptive and innate immune responses, antibiotics administration induced a large perturbation in the blood metabolome of young adults receiving inactivated seasonal influenza vaccine, including changes in bile acids, such as lithocholic acid (LCA). LCA is the most potent agonist of G protein-coupled bile acid receptor 1 (GPBAR1), which inhibits activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome, a multiprotein complex that detects pathogens and other stress-inducing signals leading to activation of innate immunity, in mice (13). Moreover, antibiotics-driven perturbation of secondary bile acids was associated with increased inflammation, including modules involved in AP-1 signaling and NLRP3 inflammasome activation. These results highlight a potential mechanism by which the microbiota can regulate secondary bile acid production and consequently inflammatory responses in humans, which can also affect vaccine responses. To what extent the dysbiosis-secondary bile acid-inflammation axis contributes to inflammaging in the elderly remains to be determined. It should also be noted that increased gut permeability, which increases with age, could result in systemic exposure to bacterial stimuli such as lipopolysaccharide, flagellin, and other pathogen-associated molecular patterns that could directly trigger up-regulation of the AP-1 and NR4A1 signaling pathways. Further studies in elderly people should thus explore the mechanisms by which gut dysbiosis modulates inflammaging and the age-associated decline in immune function known as immunosenescence.

These results could have several public health implications. Given that the impact of antibiotics on the antibody response was only evident in subjects with low baseline concentrations of antibody, it could be advantageous to receive the influenza vaccine annually to build up immune memory. A second implication concerns the potential effects of antibiotics on neonates or infants, in which immune imprinting to influenza may be minimal or nonexistent. In infant mice, antibiotics-driven earlylife dysbiosis leads to altered vaccine responses (14). A relatively sterile 9-month-old baby in utero experiences a microbial “big bang” during birth, and this microbial universe continues to expand during the first few years of life. The widespread use of antibiotics in neonates and infants raises the possibility that antibiotics-driven gut dysbiosis may exert a considerable toll on vaccination-induced immunity in the very young. Furthermore, antibiotics-driven microbiota disruptions in early life can have long-lasting effects on body weight in adulthood, long after cessation of antibiotics (15). It is therefore possible that antibiotics treatment during childhood could exert long-lasting effects on the immune system, perhaps through epigenetic imprinting. Future studies should thus be aimed at exploring both the short- and long-term effects of antibiotics on vaccine immunity in the very young. In addition, the microbiota-inflammaging axis in aging and its impact on vaccine immunity deserve attention.