2. Materials and Methods
2.1. Search Strategy
This review was conducted following the Preferred Reporting Items for Systematic reviews and Meta-Analyses checklist (PRISMA) [7
]. The protocol for this review could be found in the International Prospective Register of Systematic Reviews (PROSPERO) under registration number CRD42019134416.
To collect data on the cellular immune response to rabies vaccination, a thorough literature search was performed. We consulted the databases of MEDLINE, Embase, Web of Science, COCHRANE Library, and Academic Search Premier with the search strategy provided in Appendix A
. The search strategy included components for ‘rabies vaccination’ and ’cellular immune response’. Unpublished meeting abstracts were included in this review if they met the inclusion criteria.
2.2. Inclusion and Exclusion Criteria and Outcomes of Interest
We included studies that used inactivated cell-culture or embryonated egg-based rabies vaccine (CCEERV) in vivo or in human cells in vitro. A cellular immunological outcome in a human population needed to be assessed. Reviews and meta-analyses with references that contained relevant information to our questions were included as well, to identify missing studies in our initial search.
A study was excluded if it had no human component and if it only assessed serological or other non-cellular outcomes. Case reports and studies of which no full English text could be retrieved were excluded as well. Furthermore, studies published before 1983 were excluded, avoiding the inclusion of nerve tissue-based vaccines. Moreover, relevant immunological outcomes could not appropriately be assessed earlier.
Primary endpoints included all outcomes for B-cell responses, including the kinetics of the (memory) B-cell response, the quantity of (memory) B-cells, mean day of peak response, and composition of the response. Secondary endpoints included T-cell response outcomes, including the kinetics of the T-cell response, the number of T-cells, mean day of peak response, and composition of the response.
2.3. Selection Process
Two reviewers (L.A.O. and L.G.V.) independently screened the literature search results on eligibility for inclusion in this review. In the case of exclusion, the reason was documented. Unanimously selected studies were included in this review. Unanimously rejected studies were excluded. In case of discordance between the reviewers, the studies were reassessed by both of the reviewers, and together they concluded definitive inclusion or exclusion. Citations from review articles found in the literature search were checked to ensure that no studies were missed in the initial search. Eligible studies found in such a review, not found individually in our literature search, were included in our analysis.
2.4. Data Collection Process
One author (L.A.O.) developed a data extraction sheet and extracted relevant details. We collected the following study data: author(s), year of publication, country, study design, the total duration of follow up, type of vaccine, type of antigen for stimulation, route of administration, primary or booster vaccination regimen, and any relevant cellular immunological outcomes. Two authors (L.G.V. and J.J.M.v.D.) reviewed these extracted details.
2.5. Risk of Bias Assessment
The risk of bias was assessed using the Cochrane tool (ROBINS-I) [8
]. One of the authors (L.A.O.) assessed the bias risk for any included study. For each study, the risk of bias was reported with a severity score, as determined by the Cochrane tool. The overall risk of bias was classified as the highest bias classification in any domain for a particular study. This assessment was reviewed by two other authors (L.G.V. and J.J.M.v.D.). General publication bias might have occurred, given the limited availability of sufficiently specific tests for detecting cellular outcomes, possibly resulting in unpublished studies in which no associations were found.
This study provided an overview of the composition and kinetics of the primary and secondary T- and B-cell responses to rabies vaccine in humans. Over the past thirty-six years, the cellular response to rabies vaccine has not been studied extensively. Evidence does, however, point in the direction of an important role for B- and T-cellular immunity in response to rabies vaccination, in addition to the well-known serological parameters
The fact that the rabies vaccine is a neoantigen for almost all people creates the possibility to study the kinetics and dynamics of primary and secondary cellular immune responses to vaccination under different conditions in controlled research settings.
The primary response to the rabies vaccine was mediated by CD4+ T cells. A CD4+ T-cell subset is an essential group of cells for an adequate immune response against rabies, as was shown by the large numbers of cells in this subset responding to antigen stimulation. This lymphocyte proliferation response was already detectable from day 3 onwards. After incubation with rabies vaccine antigen, a peak in the proliferative lymphocyte response (mainly CD4+ T-cells) was reported on day 8.
B-lymphocyte subsets displayed a peak at different time points. Plasma cells peaked on day 10 after primary vaccination, with a detection window from day 7 to 14. Memory B-cells were detectable from day 10 up to at least day 28. These peaks occurred later than the T-cell response peaks, which is a logical consequence of the T-cell dependency of this response
When B-cell kinetics were compared to the antibody response kinetics, the plasma cell peak was observed on day 10 after primary vaccination, but an increase in antibody titers usually occurred later. This showed that B-cell quantities were indeed a different parameter, that might be associated with, but was not replaceable by serological outcomes.
When a booster vaccination was applied, NK cells were the first cells that could be detected. NK cells, which are responsible for early IFN-γ production after stimulation by IL-2 produced by antigen-specific T-cells, were detectable from 12 h after revaccination. Moreover, they played a role in the early cytotoxic response as the main perforin producers up to 12 h after revaccination. CD8+ cells did not seem to play a major role in the cellular immune response to the rabies vaccine, as their relative numbers even tend to decrease after vaccination. They were, however, responsible for the ‘late’ (from 12 h onwards) perforin cytotoxicity after revaccination.
On day 7 after revaccination, all studied individuals showed a positive proliferation response for one or multiple rabies vaccine antigens. This showed that antigen-specific T-cells could produce an adequate response when boosted.
For the B-cell response, it was shown that the plasma and memory B-cell response occurred in great magnitude and speed after booster vaccinations. Furthermore, IgG and IgA B-cells were detectable at much higher quantities after booster vaccinations than after primary vaccination. This goes to show that booster vaccinations enhance the B-cell immune response.
Cytokine responses appeared to increase with an increasing amount of added rabies antigen. High IL-4 and IFN-γ levels were significantly associated with high RVNA titers.
The presence of anti-rabies antibodies enhanced the T-cell response as well. However, another study showed a non-significant association between low lymphocyte proliferation indices and high RVNA titers. Information on this topic is thus still inconclusive.
We performed a systematic review of the extensive literature on cellular responses to the rabies vaccine. Multiple literature databases were used, and an exhaustive literature search was performed, ensuring that no relevant articles would be overlooked. The qualitative synthesis that resulted provides a general overview of the kinetics and dynamics of B- and T-cellular immune responses.
Several limitations, however, need to be discussed. First, the extracted studies were relatively dominated by older publications using less advanced immunological assays, which limited the translation of the findings into a more detailed understanding of rabies vaccine immunology. Secondly, the extracted studies were highly heterogeneous, using different vaccine regimens, administration routes, immunological assays, and endpoints, making comparing studies extremely difficult. Thirdly, most studies had a small study population. which hampered finding correlations between cellular responses and antibody levels. Furthermore, one should be careful when interpreting these single, small-scale studies. Finally, we used the rather broad outcome measure ‘cellular immune response’, which might have biased our review and resulted in articles being incorrectly excluded. We have tried to account for this by having two reviewers review all results of the literature search independently.
In conclusion, a general pattern in the rabies-specific T- and B-cell immune response could be identified, but the lack of homogeneity among the studies hindered a meta-analysis. In any case, this review shows that cellular parameters can be assessed detailed enough to discriminate between individuals and that they indeed do differ between individuals. Serological responders appear to be a highly heterogeneous group from a B- and T-cellular perspective. Individuals tend to respond to different antigens and have different proliferation and cytokine production profiles. Kinetic parameters that were used in the studies that we described provide a rationale for timepoints and outcome measures in further research.
Besides, the described rabies-specific kinetics provide grips for more rational, evidence-based vaccination schemes. As was shown, memory B-cells were detectable only from day 10 onwards. Therefore, revaccination at day 3 or 7 in an immunization schedule may not contribute as much as expected to the memory response and long-term protection, and another time point (for example day 10) could be a more logical choice when new vaccination regimens are implemented.
Although T-cell proliferative responses to rabies vaccine challenges have been described to a relatively greater extent, B-cell responses can still be described in more detail with new techniques. Many more cellular parameters could be used to assess the immunogenicity and predict the (long-term) protection for a vaccine. Future research could look into how NK cell, B-cell, T-cell, and antibody kinetics relate to each other in primary and secondary responses under specific conditions. If future studies can connect long-term (serological) immunity to cellular and humoral parameters, this could provide the field of rabies immunology and general vaccinology with individual prediction models. For now, there are many opportunities in this field that are still to be explored.