Seroprevalence of antibodies to measles, mumps, and rubella, and serologic responses after vaccination among human immunodeficiency virus (HIV)-1 infected adults in Northern Thailand

Background After the global implementation of national immunization programs for prevention of measles, mumps, and rubella (MMR), the prevalences of protective antibodies to these viruses are high in general population. However, there are limited data among human immunodeficiency virus (HIV)-1 infected individuals. This study aimed to determine the seroprevalence of antibodies to these viruses, and the serologic responses after vaccination among HIV-infected adults in Northern Thailand. Methods A cross-sectional study was conducted in 500 HIV-infected adults, aged 20–59 years, receiving combination antiretroviral therapy, CD4 cell count ≥200 cells/mm3, and plasma HIV-1 RNA <50 copies/mL, and 132 HIV-uninfected controls, aged 20–59 years, at Chiang Mai University Hospital during July and August 2011. Prevalences of protective antibodies to these viruses as well as serologic responses after MMR vaccination in those without protective antibody to at least one of the three viruses were compared between groups. Results The prevalences of protective antibodies to measles, mumps, and rubella were 94.2, 55.0, and 84.6 % among HIV-infected adults, and 97.7, 67.5, and 89.4 % among HIV-uninfected controls, respectively. The prevalence of protective antibody to mumps was significantly lower in HIV-infected adults (p-value = 0.010). MMR vaccination was done in 249 HIV-infected and 46 HIV-uninfected controls; at week 8 to 12 after vaccination, the seroprotective rates against measles, mumps, and rubella in HIV-infected adults were 96.4, 70.7, and 98.0 %, respectively, whereas those in HIV-uninfected controls were 100, 87, and 100 %, respectively. No serious adverse effects were observed. Conclusions In contrast to measles and rubella, the prevalence of protective antibody to mumps was low in both HIV-infected adults and HIV-uninfected controls in northern Thailand. The seroprotective rates after MMR vaccination in both groups were considerably high, except only for mumps. Therefore, MMR vaccination should be considered in all HIV-infected adults receiving antiretroviral therapy with undetectable plasma HIV-1 RNA and CD4 cell count ≥200 cells/mm3. Trial registration ClinicalTrials.gov: NCT02724852, registered on March 31, 2016.


Background
Measles, mumps, and rubella have long been regarded as diseases of childhood [1][2][3]. However, when infections from these viruses occur in adults, they are often more severe [1][2][3]. The serious complications may occur particularly in pregnant women [2]. The incidence of measles, mumps, and rubella has decreased dramatically in countries where measles, mumps, and rubella (MMR) vaccination is integrated into the national immunization program, including Thailand [4][5][6]. The first dose of measles vaccination in children aged 9-12 months and the second dose in first grade students were incorporated into the Thai national immunization program in 1984 and 1996, respectively. Rubella vaccination in first grade students was incorporated into the program 1993. The vaccine against single virus was replaced by MMR vaccine in 1997 [7]. The Thai national survey in 2003 and 2004 showed that the coverages of the first dose of measles vaccine among children aged 9-12 months and MMR vaccine among the first grade students were 96 and 94 %, respectively [8,9]. In general, measles vaccination produces protective antibody in 88-95 % of children [10,11]. The seroprevalence study among Thai population in 2004 revealed that 81 % (95 % CI 78. 8-83.5) of 1092 serum specimens tested, 89 % (95 % CI 86. .0) of 899 specimens tested, and 82 % (95 % CI 78.9-84.0) of 911 specimens tested had protective antibody to measles, mumps, and rubella, respectively [6]. Although the vaccine coverage and the rate of protective antibody to these viruses are quite high in Thai population, periodic outbreaks have been reported and might be attributed to the waning of antibody after a long period of time [12].
A study in Thai HIV-infected children receiving combination antiretroviral therapy (cART) reported that the prevalence of protective antibody to measles was only 42 % [13]. A study among HIV-infected adults in the United States reported that the prevalences of protective antibodies to measles, mumps, and rubella were 67, 91, and 95 %, respectively [14]. MMR revaccination has been studied in Thai children aged over 5 years and had CD4 > 15 % for at least 3 months after receiving cART. The study showed that the seroprotective rates against measles, mumps, and rubella declined overtime after vaccination [15]. In adults, a study in Mexico showed the early loss of measles antibody after 24 months of vaccination despite receiving cART [16]. These evidence suggested that loss of antibody responses may occur in HIV-infected people after vaccination, particularly in advanced diseases.
Up to date, there have been limited data regarding the prevalences of protective antibodies to measles, mumps, and rubella among Thai HIV-infected adults. We, therefore, conducted this study to determine 1) the prevalences of protective antibodies to measles, mumps, and rubella in HIV-infected adults and HIV-uninfected controls in northern Thailand, 2) the serologic responses at 8 to 12 weeks, and 48 weeks after MMR vaccination in those without protective antibody to at least one of the three viruses, and 3) adverse effects of MMR vaccination.

Study design, and population
The first phase of the study was a cross-sectional study to determine the prevalences of MMR-specific antibodies in HIV-infected adults and HIV-uninfected controls. HIV-infected adults receiving care at the HIV clinic of the Chiang Mai University Hospital between July and August 2011 and met the following criteria were consecutively enrolled: 1) 20-59 years old, 2) receiving cART, 3) CD4 cell count ≥200 cell/mm 3 within 6 months before enrolment, 4) plasma HIV-1 RNA <50 copies/mL, and 5) ability to provide written informed consent. HIV-uninfected controls, aged 20-59 years, were recruited from their relatives, hospital visitors, and hospital personnel, during the same period. Participants with following conditions were excluded: 1) pregnancy or lactating, 2) receiving cancer treatment, organ transplantation, ≥0.5 mg/kg/day of prednisolone or equivalent, or immunomodulating treatment, 3) impaired renal function (creatinine clearance <30 mL/ min), and 4) impaired liver function as defined by Child-Pugh C.
All participants who had no protective antibody to at least one of the three viruses were recruited into the second phase of the study. In this phase, participants were vaccinated with a single dose of MMR vaccine (GlaxoSmithKline Biologicals) at deltoid region by WK. Each 0.5 ml of vaccine contained at least 1000 TCID50 of Schwarz measles strain, at least 1000 TCID50 of RIT 4385 mumps, and at least 1000 TCID50 of Wistar RA 27/3 rubella strains. Antibodies to MMR were measured at week 8 to12, and week 48 after vaccination.

Sample size calculation
Based on a previous study in Thai adults aged ≥25 years, the prevalences of seroprotective antibody to measles, mumps, and rubella were 90.6-96.3 %, 86.2-94.9 %, and 85.6-96.9 %, respectively [6]. We estimated that the prevalence of protective antibody to each of these viruses was 80 % among HIV-infected adults and 94 % among HIVuninfected controls. For the first phase of the study, a sample size of 103 per group was required to significantly detect differences between them, at the power of 80 % using a cutoff for a two-sided alpha level of 0.05. However, we over-enrolled participants in both groups in order to have adequate number of participants without protective antibody for the second phase of the study.

Data collection
Demographic and clinical data were collected using a preprinted data collection form including age, sex, recall of prior measles, mumps, or rubella vaccination, anti-measles IgG antibody level, anti-mumps IgG antibody titer, and anti-rubella IgG antibody level at the time of enrolment. The levels of antibody to the three viruses were also measured at 8 to 12 weeks, and 48 weeks after vaccination in those who were enrolled into the second phase of the study. In addition, CD4 cell count and plasma HIV-1 RNA within 6 months before enrolment were collected for all HIVinfected adults. Local and systemic adverse effects of the vaccine were also collected.

Measurement of antibody levels to measles, mumps, and rubella
Laboratory tests were performed at the National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Bangkok, Thailand. The indirect ELISA using Enzyg-nost® Anti-Measles Virus/IgG, Anti-Parotitis-Virus/IgG, Anti-Rubella-Virus-IgG (Siemens, Marburg, Germany) were performed following the manufacturer's instructions. The optical density (OD) readings were interpreted as negative, equivocal, and positive if the delta OD was <0.1 (cut off), 0.1-0.2, and >0.2, respectively. Positive delta ODs were then converted to international units, as described in the package insert [17][18][19]. Protective antibody levels were defined as an antibody level ≥320 mIU/mL, antibody titer ≥1:500, and antibody level ≥10 IU/mL for measles, [10] mumps, [20] and rubella, respectively [21].

Statistical analysis
Data were presented in mean ± SD, median and IQR, number (%) as appropriate. Continuous variables were compared using Student's t-test or Mann Whitney-U test, and categorical variables were compared using Chi-2 test and Fisher's exact test. Statistical significance was set as 2-tailed, with pvalue of <0.05. The Bonferroni correction was used for multiple comparisons. All statistical analyses were performed using Stata statistical software version 11.0 (Stata Statistical Software: Release 11.0, Stata Corporation, College Station, TX, 2009).
The study was approved by the Faculty of Medicine, Chiang Mai University Ethical Committee. The trial was registered to the ClinicalTrials.gov (NCT02724852) on March 31, 2016.
The prevalences of protective antibodies to measles, mumps, and rubella and the antibody levels across age groups are shown in Tables 2 and 3, respectively. Among HIVinfected adults, the prevalence of protective antibody to measles was significantly lower among adults aged 20-29 years than other age groups (p-values < 0.05). There were no differences of the prevalence of protective antibody to mumps across age groups. The prevalence of protective antibody to rubella was lower among adults aged 40-49 years than aged 30-39 (p-value = 0.016), and 50-59 years (p-value = 0.035). However, overall, there were no differences in prevalences of protective antibodies to all three viruses across all age groups. Among HIV-uninfected controls, there were also no differences in prevalences of protective antibodies to all three viruses across all age groups. However, we observed a higher anti-mumps IgG titer among those aged 50-59 years than all other age groups (p-values < 0.05).
Although the prevalence of protective antibody to mumps was lower in HIV-infected adults than in HIVuninfected controls (p-value = 0.010), there were no differences in the prevalences of protective antibodies to all three viruses between HIV-infected adults and HIV-uninfected controls in all age groups. The antibody levels against measles were significantly higher in HIV-uninfected controls than HIV-infected adults in those aged 30-39 years. The antibody titers against mumps and the antibody levels against rubella were not different between HIV-infected and HIV-uninfected controls in all age groups.

Serologic responses after vaccination
There were 269 HIV-infected adults and 51 HIVuninfected controls who had no protective antibody to at least one of the three viruses. Two-hundred and fortynine HIV-infected adults and 46 HIV-uninfected controls were recruited into the second phase of the study. (Fig. 1 Table 4. Among HIV-infected adults, 11, 83, and 30 % had no protective antibodies against measles, mumps, and rubella, respectively. For HIV-uninfected controls, 5, 65 and 24 % had no protective antibodies against measles, mumps, and rubella, respectively. After vaccination, overall, there were no differences between HIV-infected adults and HIV-uninfected controls in the seroprotective rates against all three viruses. For mumps, the seroprotective rates were lower among HIVinfected adults at baseline (p-value = 0.005), at 8-12 weeks (p-value = 0.022), and at 48 weeks after vaccination (p-value = 0.061). For those who had no protective antibody at baseline, the seroprotective rates was higher among HIVuninfected controls than HIV-infected adults at 8-12 weeks (p-value = 0.007) and 48 weeks after vaccination (p-value = 0.039).

Discussion
This study demonstrated that in northern Thailand, the prevalence of seroprotective antibody to mumps in healthy adults was significantly lower than those to measles and rubella. These findings are similar to those in the study among healthy adults from northern Greece where the prevalence of seroprotective antibody to mumps was lower than that to measles [22]. However, our findings are different from those in the study among Thai adults in which the prevalences of seroprotective antibodies to all three viruses were comparably high [6]. Among HIV-infected adults, we found that the prevalence of seroprotective antibody as well as the level of antibody to mumps were significantly lower than those to measles and rubella. These findings are similar to those in studies among HIV-infected children in Thailand, [15] HIV-infected adults in England [23] and Austria, [24] but in contrast with findings in the study from the US where the prevalence of seroprotective antibody to measles was the lowest [14]. The differences among studies might be explained by the difference in the national vaccination program and also the prevalence of  natural infection in each community. In Thailand, as previously described, measles vaccine and rubella vaccine were incorporated to the national vaccination program in 1984 and 1993, respectively, while MMR vaccine replaced vaccines for single virus in 1997. Therefore, the prevalence of seroprotective antibody to mumps would be lower than those for measles and rubella. Interestingly, a study among Thai population in 2004 revealed that the prevalence of seroprotective antibody to mumps was more than 80 %, and comparable to those to measles and rubella; this high prevalence of antibody to mumps corresponded with the transmission of mumps virus during that period [6,12]. We found that there were no differences in the prevalences of seroprotective antibodies to all three viruses across all age groups for both HIV-infected adults and HIV-uninfected controls. In addition, there was no difference in the prevalences of seroprotective antibodies to all three viruses between HIV-infected adults and HIV-uninfected controls in each age group. However, this must be interpreted with caution from the small number of participants in each age group.
In the second phase of our study to determine the serological responses to MMR vaccination in participants who had no protective antibody to any of the three viruses, we found that the prevalence of seroprotective antibody to measles at baseline were very high in both HIV-infected adults and HIV-uninfected controls. Therefore, minority of them required measles vaccination. Among HIVinfected adults, the seroprotective rate against measles and mumps at 8 to 12 weeks after MMR vaccination was lower among those who had no protective antibody at baseline compared to those who had protective antibody at baseline (p-value < 0.001). The immunity to measles and mumps persisted through week 48 after vaccination for those who had protective antibody at baseline but not for those who had no protective antibody at baseline. These findings are similar to the studies among HIVinfected Thai children and HIV-infected adults in Mexico [15,16]. However, we found that the seroprotective rate against rubella after MMR vaccination was almost 100 % and the immunity to rubella persisted through week 48. The excellent antibody response to the rubella component of MMR vaccine has been shown in several studies among   * p-value = 0.005, ** p-value = 0.022, *** p-value = 0.061, **** p-value = 0.007, ***** p-value = 0.039 children [11,15]. Our study found that most of the adverse reactions from MMR vaccine were mild and selflimited. None experienced serious adverse events. Our study had some limitations. This study was not a pairmatched case-control study; therefore, there were some differences in baseline demographic characteristics between groups. Compared to the HIV-infected adults, the HIVuninfected controls in our study were younger and had more female; however, there were no significant differences in the prevalences of seroprotective antibodies at baseline as well as seroprotective rates to all three viruses after MMR vaccination among different age groups and genders (data not shown). In addition, we also found the imbalance in history of measles, mumps, or rubella vaccination, and a recalled history of infections due to those viruses between groups. However, this imbalance had no effect to the results since there were no correlations between history of vaccination or recalled history of infections due to those viruses with the prevalences of seroprotective antibodies at baseline as well as seroprotective rates to those viruses after MMR vaccination (data not shown).

Conclusions
This study demonstrated that in northern Thailand, the majority of HIV-infected adults had protective antibody to measles which is the most contagious virus among the three virus components of MMR vaccine and can cause serious complications. Lack of immunity against mumps was more prevalent in our study. Seroprotective rates against measles and mumps after MMR vaccination were lower and the immunities persisted shorter in those who had no protective antibody at baseline. Since the MMR vaccine is safe and the history of prior vaccination or infections are unreliable, MMR vaccination should be considered in all HIV-infected adults receiving antiretroviral therapy who achieve undetectable plasma HIV-1 RNA and CD4 cell count ≥200 cells/mm 3 .

Ethics approval and consent to participate
The study was approved by the Faculty of Medicine, Chiang Mai University Ethical Committee. Written informed consent was obtained before enrolment.