Evaluating Measles Vaccines: Can we Assess Cellular Immunity?

Saturday, 15th of September 2012

 

• EVALUATING MEASLES VACCINES: CAN WE ASSESS CELLULAR IMMUNITY?

Rory D de Vries and Rik L de Swart^*

* Author for correspondence

Expert Review of Vaccines

Measles remains an important cause of childhood mortality, and global eradication of the disease is being seriously considered. Because of limitations of the current live-attenuated vaccines, new vaccines and routes of administration are being investigated. In the article under review, the authors have measured measles-specific humoral and cellular immune responses after two doses of live-attenuated measles vaccine and found limited correlation between the two. This study highlights an important issue, namely that we cannot assume humoral and cellular immune responses to go hand in hand. However, it remains to be determined if assays with peripheral blood lymphocytes can be used as a correlate of protection from disease.

Live-attenuated measles virus (MV) vaccines were first introduced in the 1960s and have an impressive record of safety and effectiveness. Vaccination induces both humoral and cellular immunity, and high coverage in two-dose regimens has successfully interrupted endemic MV transmission in many geographic regions. MV-specific virus-neutralizing (VN) antibodies been identified as a correlate of protection from disease. However, several studies have suggested that cellular immune responses may also confer protection from measles in the absence of VN antibodies. Recently, Jacobson et al. measured humoral and cellular immune responses in a large cohort study of 764 subjects aged 11–22 years who had received two doses of measles-containing vaccine and demonstrated that the outcomes of these assays are not controlled or one only poorly correlated [1]. They concluded that specific humoral and cellular responses to measles vaccination are independent and suggested that both should be measured in clinical studies evaluating new measles vaccines or vaccination strategies.

VN antibody levels above 120–200 mIU/ml have been identified as a correlate of protection from measles in two independent studies [2,3]. Indeed, passive immunization with antibody preparations results in protection against measles, and maternal antibodies protect infants during the first months of life. In this respect, it is important to discriminate between protection from and clearance of MV: it is generally accepted that for the latter, cytotoxic T-cell responses are of crucial importance [4–6], as also illustrated by the fact that subjects with cellular immunodeficiency usually develop severe and often fatal disease after MV infection [7].

It has been demonstrated that some MV-vaccinated subjects with low VN antibody titers have detectable MV-specific T lymphocytes in their peripheral blood [8,9]. In addition, it was shown that MV-vaccinated subjects with undetectable MV-specific VN antibodies are still significantly protected from measles as compared with nonvaccinated subjects [3]. The incubation time of measles is relatively long, allowing the immune system to develop a secondary immune response. This may limit MV replication and restrict or completely prevent subsequent clinical disease [2,10,11].

In the study under review, the authors set out to reproduce their previous observations of an apparent independence between humoral and cellular immune responses to MV vaccination [12]. In comparison with their previous report, they now used a larger study cohort and improved immunological assays.

Summary of methods & results 

Study cohort

The study cohort consisted of 764 individuals, and was a combination of two previously described cohorts [13,14]. The first included 388 healthy children (11–19 years) and the second one had 376 healthy children and young adults (11–22 years). All subjects had received two doses of measles vaccine, the first one at approximately 12 months and the second one at least 1 month after the first dose.

Humoral immune responses

In their previous study, the authors used MV IgG ELISA assays to assess antibody levels to measles. Here, MV-specific VN antibody levels were determined by using a plaque reduction microneutralization (PRMN) assay [15]. Briefly, serial dilutions of heat-inactivated serum were mixed with 20–60 plaque-forming units of recombinant MV expressing green fluorescent protein, incubated for 1 h at 37°C and subsequently plated with Vero cells in a 96-well plate. After 2 days, plaques were counted, 50% end point titers were determined and titers were converted to international units per milliliter using the Third International Standard for Anti-Measles.

Cellular immune responses

In their previous study, the authors used lymphocyte proliferation assays to assess cellular immunity to measles. In this study, they used an IFN-γ ELISPOT assay, for which they previously reported high accuracy and precision [16]. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in the presence or absence of live MV vaccine strain Edmonston B (multiplicity of infection: 0.5) or stimulated with phytohemagglutinin as positive control. After determining the optimal counting parameters, ELISPOT plates were analyzed automatically. In addition, the secretion of 12 different cytokines in the supernatants of MV-stimulated PBMCs was used as a secondary measure of cellular immunity. Results for seven cytokines (IL-2, IL-6, IL-10, IFN-α, IFN-γ, IFN-λ1 and TNF-α) were analyzed. IL-4, IL-5, IL-12, IL-13 and IL-17 were excluded, since the authors did not detect MV-specific production of these cytokines.

Study objective

As a primary objective, the authors assessed correlations between MV-specific humoral and cellular immune responses, testing their hypothesis that these responses following a second dose of measles-containing vaccine are independent.

Results

The authors report a median PRMN titer of 844 mIU/ml, and 68 subjects (8.9%) had antibody levels below the threshold for protection from disease. Furthermore, a median of 36 IFN-γ spot-forming cells per 2 × 10⁵ PBMCs was detected. Using Spearman’s rank correlation test, the authors found only a weak and even a negative correlation between PRMN titers and IFN-γ ELISPOT counts. No quantitatively important correlations between PRMN titers and cytokine levels were observed. The only substantial correlations found were between cytokine levels: IL-2 levels were positively associated with IL-10 and IFN-γ, IL-10 with IFN-α and IFN-γ and IFN-α with IFN-λ1. Correlations did not change when controlling for demographic and clinical variables. Multiple linear regression analysis demonstrated that all markers for cellular immunity taken together were significantly associated with PRMN levels, but explained only 4.7% of the variability in the VN antibody titers. 

Discussion & significance of results

This study confirms previous observations that MV-specific antibody levels and T-cell responses detected in the peripheral blood of vaccinated subjects do not correlate. In their discussion, the authors recognize they cannot exclude that some individuals may have been exposed to wild-type MV. Furthermore, blood samples were collected months to years after the second vaccination, so results may have been clouded due to waning immunity. However, the large number of subjects included in the study, the statistical evaluation based on median rather than mean responses and the fact that the subjects resided in an area where virtually no measles cases were observed during the study period strongly suggest that their conclusions are justified. The authors also conclude that in future measles vaccination studies both humoral and cellular immune responses should be measured to demonstrate non-inferiority of candidate new vaccines or vaccination routes in comparison with the existing measles vaccines.

Although seemingly justified, some problems are associated with this statement. As described above, VN antibodies have clearly been identified as a correlate of protection against measles. Moreover, it is thought that VN assays measure the actual biological function of MV-specific antibodies: in subjects who carry protective levels of VN antibodies the virus is neutralized in vivo in a similar way as measured in the VN assay in vitro. Although it is generally accepted that cellular immunity indeed contributes to protection from measles [17], two crucial problems need to be solved:

	• Can biological function of MV-specific memory T lymphocytes be measured?
	• Is it possible to use PBMCs for this purpose?

IFN-γ ELISPOT assays can be reliably used to determine the frequency of MV-specific T cells in peripheral blood. However, measurement of the production of IFN-γ upon in vitro stimulation is not necessarily directly related to the function these cells exert in vivo. In order to generate an effective secondary immune response, clonal expansion of specific memory T lymphocytes is a probable first response upon exposure to MV. This response is actually mimicked in the ‘old-fashioned’ lymphoproliferation assays. In recent years, several assays have been developed to measure the capacity of virus-specific T lymphocytes to control virus replication or clear infected cells [6,18,19], but it remains to be determined if any of these assays indeed measure the postulated protective T-cell responses.

Another fundamental problem is that it is becoming increasingly clear that virus-specific memory lymphocytes may not circulate but rather reside in mucosal or peripheral tissues [20]. If these are the cells that confer protection in subjects vaccinated against measles with nondetectable VN antibody levels, it may not be possible to design functional assays using PBMCs as correlates of protection.

Five-year view

 we expect further development and standardization of assays for the measurement of MV-specific cellular immune responses. Most importantly, there is an urgent need to apply and validate these assays in measles outbreak studies; it may be possible to collect PBMCs from vaccinated infants before exposure during these studies. Such studies would be able to address the questions raised earlier.

Key issues

	• Live-attenuated measles vaccines are safe and effective, and induce both humoral and cellular immune responses.
	• Implementation of high vaccination coverage in a two-dose schedule has resulted in regional elimination of measles in many parts of the world.
	• Measles virus (MV) is highly contagious, and drops in vaccination compliance quickly result in reintroduction of the virus leading to small or large measles outbreaks.
	• MV-specific virus-neutralizing antibodies as measured by plaque reduction neutralization assays have been identified as a correlate of protection from measles.
	• The contribution of cellular immune responses to long-term protection is poorly understood, but it has been demonstrated that vaccinated seronegative subjects can be protected from measles.
	• Although cellular immune responses have been investigated in individuals who had recovered from acute measles, a study into the correlates of protection has never been performed.
	• The study reviewed here confirms previous observations of the independence of MV-specific humoral and cellular immune responses after two doses of MV vaccine.
	• Considering that memory T lymphocytes may reside in peripheral and/or mucosal tissues, it remains to be determined if cellular immunological correlates of protection from measles can ever be identified using peripheral blood lymphocytes.

 

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter discussed in the manuscript. This includes employment, consultancies, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this article.

 

References

Top of Form ChooseTop of pageABSTRACTSummary of methods & resu...Discussion & significance...Five-year viewReferences << Bottom of Form

 

1	Jacobson​‌ RM, Ovsyannikova IG, Vierkant RA, Pankratz VS, Poland GA. Independence of measles-specific humoral and cellular immune responses to vaccination. Hum. Immunol.73,474–479(2012). [CrossRef] [CAS]
2	Chen​‌ RT, Markowitz LE, Albrecht P et al. Measles antibody: reevaluation of protective titers. J. Infect. Dis.162(5),1036–1042(1990). [CrossRef] [Medline] [CAS]
3	Samb​‌ B, Aaby P, Whittle HC et al. Serologic status and measles attack rates among vaccinated and unvaccinated children in rural Senegal. Pediatr. Infect. Dis. J.14(3),203–209(1995). [CrossRef] [CAS]
4	Permar​‌ SR, Moss WJ, Ryon JJ et al. Prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction. J. Infect. Dis.183(4),532–538(2001). [CrossRef] [Medline] [CAS]
5	Permar​‌ SR, Klumpp SA, Mansfield KG et al. Role of CD8(+) lymphocytes in control and clearance of measles virus infection of rhesus monkeys. J. Virol.77(7),4396–4400(2003). [CrossRef] [CAS]
6	de Vries​‌ RD, Yüksel S, Osterhaus ADME, de Swart RL. Specific CD8(+) T-lymphocytes control dissemination of measles virus. Eur. J. Immunol.40(2),388–395(2010). [CrossRef] [CAS]
7	Permar​‌ SR, Griffin DE, Letvin NL. Immune containment and consequences of measles virus infection in healthy and immunocompromised individuals. Clin. Vaccine Immunol.13(4),437–443(2006). [CrossRef] [CAS]
8	Ward​‌ BJ, Boulianne N, Ratnam S, Guiot MC, Couillard M, De Serres G. Cellular immunity in measles vaccine failure: demonstration of measles antigen-specific lymphoproliferative responses despite limited serum antibody production after revaccination. J. Infect. Dis.172(6),1591–1595(1995). [CrossRef] [Medline] [CAS]
9	Bautista-López​‌ N, Ward BJ, Mills E, McCormick D, Martel N, Ratnam S. Development and durability of measles antigen-specific lymphoproliferative response after MMR vaccination. Vaccine18(14),1393–1401(2000). [CrossRef] [CAS]
10	Muller​‌ CP, Huiss S, Schneider F. Secondary immune responses in parents of children with recent measles. Lancet348(9038),1379–1380(1996). [CrossRef] [Medline] [CAS]
11	Helfand​‌ RF, Kim DK. Gary HE Jr et al. Nonclassic measles infections in an immune population exposed to measles during a college bus trip. J. Med. Virol.56(4),337–341(1998). [CrossRef] [CAS]
12	Dhiman​‌ N, Ovsyannikova IG, Ryan JE et al. Correlations among measles virus-specific antibody, lymphoproliferation and Th1/Th2 cytokine responses following measles–mumps–rubella-II (MMR-II) vaccination. Clin. Exp. Immunol.142(3),498–504(2005). [CAS]
13	Haralambieva​‌ IH, Ovsyannikova IG, Kennedy RB et al. Associations between single nucleotide polymorphisms and haplotypes in cytokine and cytokine receptor genes and immunity to measles vaccination. Vaccine29(45),7883–7895(2011). [CrossRef] [CAS]
14	Ovsyannikova​‌ IG, Haralambieva IH, Vierkant RA, Pankratz VS, Jacobson RM, Poland GA. The role of polymorphisms in Toll-like receptors and their associated intracellular signaling genes in measles vaccine immunity. Hum. Genet.130(4),547–561(2011). [CrossRef] [CAS]
15	Haralambieva​‌ IH, Ovsyannikova IG, Vierkant RA, Poland GA. Development of a novel efficient fluorescence-based plaque reduction microneutralization assay for measles virus immunity. Clin. Vaccine Immunol.15(7),1054–1059(2008). [CrossRef]
16	Ryan​‌ JE, Ovsyannikova IG, Poland GA. Detection of measles virus-specific interferon-gamma-secreting T-cells by ELISPOT. Methods Mol. Biol.302,207–218(2005). [CAS]
17	Amanna​‌ IJ, Slifka MK. Contributions of humoral and cellular immunity to vaccine-induced protection in humans. Virology411(2),206–215(2011). [CrossRef] [CAS]
18	van Baalen​‌ CA, Gruters RA, Berkhoff EG, Osterhaus ADME, Rimmelzwaan GF. FATT-CTL assay for detection of antigen-specific cell-mediated cytotoxicity. Cytometry. A73(11),1058–1065(2008). [Medline]
19	Kreijtz​‌ JHCM, de Mutsert G, van Baalen CA, Fouchier RAM, Osterhaus ADME, Rimmelzwaan GF. Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus. J. Virol.82(11),5161–5166(2008). [CrossRef] [Medline] [CAS]
20	Sheridan​‌ BS, Lefrançois L. Regional and mucosal memory T cells. Nat. Immunol.12(6),485–491(2011). [CrossRef] [CAS]

Affiliations

Rory D de Vries

Department of Virology, Erasmus MC, PO Box 2040, 3000 CA, Rotterdam, The Netherlands

Rik L de Swart

Department of Virology, Erasmus MC, PO Box 2040, 3000 CA, Rotterdam, The Netherlands. r.deswart@erasmusmc.nl