Despite the availability of an effective vaccine, measles outbreaks continue to be a major global public health concern [1
]. Over 20 million measles infections occur annually; 122,000 deaths due to measles occurred worldwide in 2012 [1
]. Insufficient vaccine coverage [1
], along with primary and secondary vaccine failure [3
] and early waning immunity [9
], are contributors to the sustained occurrence of measles in developing countries and the resurgence of measles cases in developed countries [3
]. In the first six months of 2014, the US reported more measles cases (397 cases) than it has reported annually since 2000 [12
]. Many of the measles cases in the recent outbreaks have been due to failure to vaccinate, but primary and secondary vaccine failure also play a role in measles susceptibility and outbreaks [12
]. Various reports from the literature estimate 2%–10% of vaccinated individuals (with two measles-mumps-rubella/MMR doses) fail to develop a protective measles antibody response, which can result in infection upon exposure [13
]. Thus, vaccine failure and waning immunity are major concerns, and further studies of the measles vaccine’s immunogenicity and correlates of protection are necessary. In addition, an accurate and feasible method for monitoring measles vaccine-induced protective immunity (e.g., functional neutralizing antibodies relevant to protection and/or other correlates of protection) is crucial for achieving measles eradication [23
The current gold standard in measles serology is the measurement of neutralizing antibodies directed against the two measles virus (MV) surface glycoproteins—the hemagglutinin (H) and fusion (F) proteins—by the standard plaque reduction neutralization (PRN) test, or its modified high-throughput version—the fluorescence-based plaque reduction microneutralization (PRMN) assay [16
]. MV fusion to, and entry into, cells is the result of concerted efforts of the H and F proteins [25
]. Depletion of H- and F-specific antibodies from the serum of vaccinated individuals resulted in the abrogation of virus neutralizing activity, as demonstrated by de Swart et al.
]. Depletion of only H-specific antibodies almost completely abrogated neutralizing activity, while depletion of only F-specific antibodies had a minimal effect on virus neutralization titers [28
]. This suggests that H-specific antibodies are the main correlate of MV neutralization.
Although the H and F neutralizing antibodies are currently the most studied and used correlates of MV protection, their measurement is labor intensive, costly, and/or requires special equipment and trained personnel [24
]. Other MV proteins include: The nucleocapsid (N) protein, the phosphoprotein (P), and the matrix (M) and polymerase (L) proteins [30
]. In addition, the non-structural C and V proteins are expressed upon transcription of the virus in infected cells and are implicated as immune evasion factors associated with increased MV virulence [30
]. Clearly, there are several alternate humoral immune markers that could potentially serve as additional correlates of protection, but in-depth information is lacking with regard to the levels of antibodies against these proteins after MMR vaccination.
Comprehensive evaluation of measles-specific humoral immunity after vaccination is important for determining new and/or additional correlates of vaccine immunogenicity and efficacy, and for acquiring new insights into the immune effector mechanisms related to long-term protection after immunization. In this study, we performed proteomic profiling of IgG measles-specific humoral immune responses in 150 vaccine recipients (after two MMR vaccine doses) representing the extremes of the measles-specific neutralizing antibody response (75 high antibody responders and 75 low antibody responders) using proteome microarray technology (examining the entire measles virus proteome) and modeled antibody response to identify a model predicting neutralizing antibody titer [36
]. This information has the potential to lead to the development of more effective and feasible methods for evaluating protective immunity after measles vaccination.
We and others have discussed the use of the classical PRN assay and its high-throughput alternative, the PRMN assay, as “gold standard” assays to monitor neutralizing antibody response and protective functional immunity after measles vaccination and/or infection [16
]. Unlike the quick and accessible enzyme immunoassays (EIA), including EIAs, which detect anti-N antibodies (the antibodies formed most abundantly in response to infection and immunization) [24
], PRN/PRMN assays assess the neutralizing anti-H and anti-F antibodies that prevent a cytopathic effect and plaque formation on cell monolayers by measuring the serum dilution capable of preventing 50% of plaque formation by MV (50% Neutralizing dose /ND50
, PRN titer). Despite the differences in the antibodies of detection, relatively good correlation between EIA and PRN/PRMN assays has been reported [24
]. Although the neutralization assays tend to have a higher specificity, they are slow, labor-intensive and/or require highly trained personnel and specialized instrumentation [16
], so seroprevalence studies often resort to EIA for convenience.
Global profiling of humoral immune response using unbiased chip technology could materially assist high-throughput vaccine monitoring through the generation of comprehensive high-resolution snapshots of the antigen-specific immunoproteome (antibodies) and the identification of new and/or additional biomarkers for vaccine response.
The ultimate goal of our study was to use unbiased proteome microarray data in order to identify humoral factors/components and/or models that can be used to predict and/or discriminate among vaccine-induced phenotypes (protected vs. non-protected phenotype based on neutralizing antibody response) and that could serve as potential correlates of protection and biomarkers of vaccine immunogenicity and/or efficacy.
High-throughput proteome microarray technology has been successfully used to probe humoral immunity to different microorganisms, including viruses (e.g., vaccinia virus, human papillomaviruses, herpes simplex virus 1 and 2) [36
]. As expected, using this relatively new proteome approach, we were able to detect antibodies to most of the structural MV proteins, as well as the non-structural V protein (no antibodies were detected against M, C and L-s2 proteins in our study cohort). The microarray antibody reactivity measured against the MV surface glycoproteins H and F, as well as against other MV structural proteins (N, P and L-s3 comprising the 1234 to 1900 AA fragment of the MV L protein), was significantly different between the studied vaccine-induced immune phenotypes (high vs.
low measles-specific neutralizing antibody phenotype) and positively correlated with the neutralizing antibody titer, but there was no, or limited, correlation with other immune response outcomes (cytokine secretion and IFNγ ELISPOT response), similar to other studies [16
]. These data indicate that the proteome microarray does not capture the same measure of functional antibody activity as the neutralization assays.
As reviewed by Bouche et al.
], the neutralizing B cell response to measles virus has been mapped solely to the H and F proteins, in particular to the H protein conformational epitopes and, to a lesser extent, to the F protein [28
]. Antibodies to other proteins (N, P and M) were not systematically studied in larger cohorts, and the significance and contribution of anti-L, anti-V and anti-C antibodies (if any) to the measles-specific immunoproteome is unclear. It is possible that antibodies with these specificities participate in the antibody-dependent cell-mediated cytotoxicity/ADCC; alternatively, they may not be involved in immune protection.
Our study findings provide interesting insight into the global measles-specific humoral immune response in study subjects who represent the extremes of the neutralizing antibody response to measles vaccine years after the second MMR vaccination (median 7.2 years). The study demonstrates that both membrane and non-membrane viral proteins were antigenic, and elicited sustained and stable antibody response years after immunization (including the non-structural V protein in part of the study subjects). Of note, antibodies to neutralizing conformational H/F epitopes and to conformational epitopes on other MV proteins were likely underrated or not detected (although antibodies to other epitopes on these antigens were readily detected using the microarray technology) due to the E. coli-based IVTT cell-free expression system used for protein/antigen expression, which poses an important limitation to the interpretation of our results. With that in mind, we observed the highest seroprevalence of antibodies directed to the MV phosphoprotein (P) and nucleoprotein (N). Both proteins are highly expressed in infected cells and the high correlation with neutralizing antibody response may reflect more efficient measles vaccine virus replication in some individuals compared to others. Interestingly, antibodies to the large polymerase (L) MV protein (in particular, antibodies to the L-s3 portion, comprising the 1234 to 1900 AA fragment, including the second flexible hinge/H2 region (1695–1717AA) of the L protein), in addition to anti-N, anti-P and anti-F (and, to a lesser extent, anti-H) antibodies were also prevalent and correlated with neutralizing antibody response and/or were associated with—and predictive of—neutralizing antibody response in a univariable logistic regression model. The measles virus L protein harbors important functions (some in conjunction with the P protein), such as P binding, genome replication and viral mRNA synthesis (RNA capping, methylation, polyadenylation, phosphodiester bond formation) [70
], that are associated with distinct functional domains; however, information about human antibody response to this protein is missing or limited. Similarly, the literature contains only a modicum of data on the seroprevalence of human antibodies directed to MV P protein (an essential polymerase cofactor and immune evasion factor) in vaccinated individuals [58
]. It is possible that antibodies directed against N, P or other MV proteins are integral part of the MV-specific antibody-dependent cellular cytotoxicity (ADCC) and thus have a functional role in the host defense and protection [72
We analyzed the seroprevalence of antibodies to all MV proteins using a novel high-throughput proteome technology and statistical modeling, and identified diverse antibody target recognition and different serological patterns, distinguishing high from low neutralizing antibody response. In particular, we identified a multivariable logistic regression model, predictive of neutralizing antibody response, that correctly classified the vaccine-induced immunophenotypes (high or low neutralizing antibody response) 87% of the time. Collectively, high microarray antibody reactivities to MV-P, MV-N and MV-F and lack of antibody reactivity to MV-L-s1/s4 and MV-V were predictive of high neutralizing antibody response. While we have not compared the microarray profiling of antibodies to conventional EIAs, we have correlated this technology with the gold standard in measles serology for measurement of functional antibodies relevant to protection (PRN/PRMN assays). Based on our results, we can speculate that some conventional EIA assays (e.g., those using purified N or P proteins) may correlate well with the neutralizing antibody response. The benefits of the microarray technology include the high sensitivity in antibody detection (for antibodies covering the entire measles virus proteome), the quick turn-around time and feasibility for large population-based studies. Our study was designed to identify which antibodies/models best discriminate between the high and the low ends of the neutralizing antibody distribution with the goal of identifying subjects who may not respond to the measles vaccine (non-responders or low responders). This data will inform future research aimed at predicting primary and/or secondary vaccine failures after measles vaccination.
The strengths of our study include the well-characterized study cohort that includes subjects who represent the extremes (high and low) of the neutralizing antibody response after vaccination, the comprehensive immunophenotyping data and known vaccine history for our cohort (two doses of MMR vaccine) in a geographic location with no known circulating wild type virus, and the standardized QA/QC laboratory procedures and statistical modeling approach.
The limitations of our study include the constraints of the microarray technology and the antigen-expression system used limiting the detection of antibodies to conformational epitopes. While this technology is able to detect antibodies directed against epitopes based on the primary sequence (i.e.
, primary and secondary structure), antibodies to conformational epitopes dependent on disulfide bonds and/or other post-translational modification are not detected. Methods based on H and F proteins with preserved conformational epitopes (e.g., viable transfected human cells expressing H or F proteins) are attractive, but not practical for larger studies [28
]. Another limitation is the exclusion of individuals with intermediate levels of neutralizing antibodies from the study (by excluding the values that are closest to the center of the distribution, i.e.
, those with the “least weight”, the reported correlations may be higher than they would have been if these values were included). Our study design with high/low immune response groups was chosen to maximize the biological difference and to have a better power to detect differences with this sample size.
In summary, our study findings further the understanding of immune responses and long-term humoral proteome patterns to the measles component of the MMR vaccine following live viral vaccination. While the identified factors may not directly reflect the neutralization capacity of human sera, the models presented herein can be potentially used to predict neutralizing antibody response and functional protective immunity to measles vaccine and/or lay the foundations (perhaps in conjunction with markers of cellular immunity in the models) for the discovery of new/additional biomarkers of vaccine response.