The Interaction Between Measles and DTP Vaccination

The Interaction Between Live and Killed Vaccines: the Effect of the Diphtheria-tetanus-whole Cell Pertussis (DTP) Combined Vaccine on T Cell Memory Following Measles Vaccination

The purpose of this study is to investigate for the broad immunological effects of administering measles vaccine (MV) and diphtheria-tetanus-whole cell pertussis vaccine (DTP) to 9 month old Gambian infants, either alone or together. Effects on vaccine-specific immune responses, innate immunity, and immune memory were studied. The hypothesis is that when MV and DTP are given together there will be more inflammation and this will interfere with generation of immunity to the vaccine and to other non-vaccine related stimuli.

Many studies demonstrate that routine Expanded Program on Immunisation (EPI) vaccinations have "non-specific" or "heterologous" effects on child survival that cannot be accounted for by protection against the vaccine specific disease. These effects were overlooked in the past because it was assumed that the impact on survival would be proportional to reduction in the targeted infection. Large epidemiological studies in Bangladesh and Guinea-Bissau have shown that live vaccines, including measles vaccine (MV), have beneficial effects on overall mortality which cannot be explained by protection against the vaccine specific disease. Deleterious non-specific effects were shown convincingly in the high-titre MV trials in which girls, but not boys, had a two-fold increase in mortality even though the vaccine was fully protective against measles. A reinterpretation of these trials indicated that the most likely explanation was that the increased female mortality was due to inactivated vaccines provided after MV. It has further been shown in smaller observational studies that receiving diphtheria, tetanus and whole cell pertussis combined vaccine (DTwP) either with or after MV negates the beneficial effects of MV, and may lead to increased mortality. However, the increased mortality of girls in male-female twin pairs was not apparent when MV was given after the last dose of DTwP. In the present EPI schedule in The Gambia, DTwP is administered as a single dose vaccine at 2, 3 and 4 months, and MV at nine months. However, many children, particularly in low income countries, present late for their vaccines and may subsequently receive them in the wrong order. In particular DTP is often given at the wrong time point, e.g. the third DTP dose (DTP3) with MV. Given the increasing evidence that the order in which vaccines are administered is crucial, the routine practice of administering missed EPI vaccines at late presentation or boosting with DTP in year 2 may not be advisable. The mechanism behind these effects is unknown, and whether there is an immunological basis has yet to be explored. The majority of controlled trials of vaccine efficacy have focused on vaccine specific antibody (Ab) responses to test vaccine efficacy, and few studies have investigated T cell memory induction, despite the fact that it is likely to be critical for long term protection. Indeed, the cellular response to MV seems to be crucial in protecting against severe disease and death, and cellular responses are required to protect against Bordetella pertussis infection. Live vaccines such as bacillus Calmette-Guerin (BCG) and MV have been shown to stimulate Th1 type immune responses. The immune response to DTwP, a killed vaccine, tends to be Th2 biased in early life, despite the fact that the whole cell pertussis component biases towards a Th1 response. DTwP contains an aluminium based adjuvant, aluminium hydroxide, which is excellent at promoting humoral and Th2 responses, but poor at generating good cell mediated immunity and long term T cell memory. This polarisation of cellular reactivity to live and killed vaccines may explain why administering a killed vaccine with a live vaccine abrogates the beneficial effect of the live vaccine. Conversely, administering a live vaccine after DTwP may cause a shift towards protective Th1 type immunity, thus diminishing the harmful effects of DTwP and providing a non-specific survival benefit. Interestingly, aluminium hydroxide has been shown to cause enhanced susceptibility to tuberculosis (TB) in animal models. Thus the aluminium adjuvant in DTwP may play a role in the deleterious effect of DTwP on live vaccines, possibly through an influence on the generation of T cell memory to other infections and vaccines. Attributing the observed effects of the DTwP / MV interactions entirely to shifts in the T helper cell 1 (Th1) / Th2 profile is likely to be an over simplification, but provides a good starting point for unravelling this phenomenon. Other arms of the immune response that need to be considered are regulatory T cells (Tregs) and the Th17 inflammatory T cell lineage. The former are a heterogeneous group of naturally occurring and induced T cells, and have been shown to play a regulatory role in immunity to a number of infectious diseases, but almost nothing is known about their generation or functional role in vaccine immunogenicity in humans. Newborns are known to have high levels of functional Tregs, and these are likely to be essential in controlling the immune response to antigens encountered in early life, as well as modulating self reactive responses. Measles vaccination causes suppression of T cell responses, and measles vaccinated children upregulate Forkhead Box P3 (FOXP3) expression (Ota et al, personal communication), suggesting a role for Tregs. Thus Treg induction is likely to be an important component of the immune response to MV. This study will investigate in detail the immunological consequences of giving DTP or MV alone or at the same time. The study will involve an intervention trial to analyse the effect of giving DTP with MV on the generation of T cell memory (effector and central), humoral responses, pro-inflammatory cytokine profile (Th1, Th2, Th17) and Treg responses to measles and recall antigens. Our detailed immunological studies will provide vital immunological data on potential mechanisms of the DTP / MV interaction. A comprehensive understanding of the immunological effects of administering either DTP alone or DTP and MV simultaneously will help us understand why these interventions might be detrimental, and may suggest the need to refine current EPI practices. It is also crucial to gain a detailed understanding of the immunological effects of possible deleterious interactions between the current EPI vaccines, before introducing new vaccines that are becoming available for testing in early life e.g. TB and malaria vaccines. Furthermore, understanding mechanisms whereby MV provides survival benefits might identify strategies that can be harnessed in future vaccine design. All infants will be given a MV challenge at 18 months of age and immunological parameters will be analysed 1 month later. This will establish whether any vaccine group effects at 10 months off age persist until 18 months of age and influence the immune response to a new immune challenge. Two primary hypotheses as detailed below will be tested as a starting point, but other immunological assays will be conducted. Furthermore all analyses will take sex into account since the heterologous effects of vaccines are different in males and females, with females generally being more susceptible. Hypotheses: DTP (a killed vaccine) drives primarily a Th2 response and MV (a live vaccine) drives primarily a Th1 response. A DTP driven Th2 response occurring at the time of trying to activate a Th1 response to MV will interfere with the priming of measles specific memory responses and responses to other recall antigens. The extra Th2 stimulation from DTP at the time of MV will provide too many signals to allow the generation of regulatory T cells (Tregs) which are an essential component of the immunological response to MV.

Measles Vaccine at 9 months of age. Measles Vaccine at 18 months of age. All vaccines given as per normal Gambia schedule until 9 months of age, including third dose of diphtheria-tetanus-whole cell pertussis (DTP3), hepatitis B vaccine (HBV) and oral polio vaccine (OPV) at four months of age. At 9 months of age given a single standard intramuscular (i.m.) dose of measles vaccine (MV) (Edmonston Zagreb strain, Serum Institute of India Ltd., Pune, India) into the deltoid. Yellow fever vaccine (YF) and OPV administered at 11 months of age. Given a standard MV challenge at 18 months of age.

Measles Vaccine at 9 months of age. DTP Vaccine at 9 months of age. Measles Vaccine at 18 months of age. DTP3 dose withheld and given HBV and OPV at four months of age. At 9 months of age given a single standard intramuscular (i.m.) dose of measles vaccine (MV) (Edmonston Zagreb strain, Serum Institute of India Ltd., Pune, India) into the deltoid, and i.m. DTP (Serum Institute of India Ltd.) in the thigh. Yellow fever vaccine (YF) and OPV administered at 11 months of age. Given a standard MV challenge at 18 months of age.

DTP Vaccine at 9 months of age. Measles Vaccine at 18 months of age. DTP3 dose withheld and given HBV and OPV at four months of age. At 9 months of age given i.m. DTP (Serum Institute of India Ltd.) in the thigh. MV, OPV and YF administered at 11 months of age. Given a standard MV challenge at 18 months of age.

Single standard intramuscular (i.m.) dose of measles vaccine (MV) (Edmonston Zagreb strain, Serum Institute of India Ltd., Pune, India) into the deltoid at 9 months of age

Single intramuscular (i.m.) dose of diphtheria-tetanus-whole cell pertussis vaccine (Serum Institute of India Ltd.) into the thigh at 9 months of age

Single standard intramuscular (i.m.) dose of measles vaccine (MV) (Edmonston Zagreb strain, Serum Institute of India Ltd., Pune, India) into the deltoid at 18 months of age

The IFN-gamma:IL-4 cytokine ratio in plasma samples taken 4 weeks after vaccination measured by multiplex assay using the Bio-Plex 200 Suspension Array system. Ratio compared between the 3 intervention groups with the hypothesis that the MV+DTP group will have a higher ratio than the other 2 groups.

Regulatory T cell (Treg) frequencies (among all gated CD4+ T cells) at 9 and 10 months of age determined by flow cytometry and compared between the 3 intervention groups with the primary hypothesis that the MV+DTP group will have lower Treg frequencies than the MV group.

Effect of combining MV and DTP on measles antibody responses to see if combining vaccines interferes with antibody generation. Plasma measles antibodies measured by haemagglutination inhibition assay at 9 and 10 months of age.

Plasma diphtheria and tetanus toxoid antibodies were measured by multiplex microsphere-based fluorescent immunoassay at 9 and 10 months of age.

Plasma pertussis toxoid antibodies were measured by multiplex microsphere-based fluorescent immunoassay at 9 and 10 months of age

Measles antibody levels were measured by haemagglutination inhibition assay (HAI) at 19 months of age, 4 weeks after MV challenge at 18 months of age. The aim was to determine whether the vaccination schedule at 9 months of age altered responses to a subsequent MV challenge at 18 months of age i.e., the antibody measurement was made 10 months after the first study intervention.

Inclusion Criteria: Healthy 4 month old infants All EPI vaccines received to date according to current Gambia government schedule Normal weight for age according to growth chart Exclusion Criteria: Temperature >37.5°C Any history of ongoing chronic illness

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