轮状病毒（Rotavirus，RV）是导致世界范围婴幼儿重症腹泻的主要病原体之一，轮状病毒感染被称为“民主病”，无论发达还是发展中国家儿童都会感染，给世界各国带来了严重的社会和经济负担。但在发展中国家引起的5岁以下儿童死亡率更高。疫苗接种是唯一预防轮状病毒腹泻，降低死亡率的有效途径。近十年来，葛兰素史克公司生产的人类轮状病毒减毒活疫苗（Rotarix）和默沙东公司生产的口服轮状病毒五价活疫苗（RotaTeq）在世界范围广泛使用。疫苗临床试验和上市后调查数据显示轮状病毒减毒活疫苗在发达国家具有高效的保护作用，然而在最需要它们的发展中国家，疫苗的有效性和免疫原性却不理想。对于这种现象，亟待找出原因以提高轮状病毒疫苗在发展中国家的有效性。学者们针对此现象曾经进行过研究，从接种者的营养状况、肠道菌群、母乳喂养行为以及病毒株流行的不同入手，解释免疫原性差异的原因。但这些阐述综合起来分析并不能够给出全面系统合理的解释，也有待于从不同侧面进行更深入的研究。
免疫系统具有复杂的网络结构，导致轮状病毒疫苗在不同地区免疫原性和有效性不同的也可能不仅限于上述几个因素。鉴于此，本研究从接种者机体内免前母传抗体、人类白细胞抗原（Human Leukocyte Antigen，HLA）基因多态性和同时接种口服脊髓灰质炎病毒减毒活疫苗（Oral Poliovirus Vaccine，OPV）的行为三个方面分析可能影响轮状病毒减毒活疫苗的免疫反应的因素。  
本研究中，6～13周龄健康婴幼儿接种三剂III价轮状病毒基因重配减毒活疫苗，每剂间隔1个月，分别在首剂接种前、第3剂接种后1个月采集血样，进行G2、G3、G4型特异性轮状病毒免疫球蛋白A（Rotavirus-Immunoglobulin A，RV-IgA）抗体测定。分别根据各型免后RV-IgA是否阳转，将接种人群分为各型血清阳转组和血清未阳转组。同时检测接种人群的免前血清G2、G3、G4型特异性轮状病毒中和抗体水平。统计各型中和抗体阳性率和抗体水平在两组间是否有差异。结果发现：接种人群中免前3个型特异性的轮状病毒中和抗体阳性率和抗体几何平均滴度分别为 G4型最低（49.24%，12），G2型居中（82.95%，20），G3型最高（100%，91）。与此相对应地，群体免后呈现出RV-IgA的血清抗体水平G3型（76U/mL）最低，G2型（86U/mL）居中，G4型（90U/mL）最高的分布特点。免前各型特异性中和抗体的阳性率和抗体水平均为血清阳转组低于血清未阳转组，除G3型外，G2和G4型免前中和抗体的阳性率和抗体水平在两组之间均有统计学差异（P＜0.05）。提示母传抗体可能会抑制轮状病毒减毒活疫苗的免疫反应。
根据接种者首次接种III价轮状病毒基因重配减毒活疫苗时的年龄，将其分为两组，平均年龄分别为8周龄和12周龄，统计各型免前中和抗体和免后RV-IgA在两组间的差异，分析推迟免疫接种程序是否能够消除母传抗体的影响，增强疫苗免疫反应。与8周龄人群相比，在12周龄接种人群中，除G2型免前中和抗体阳性率略高（83.19%:82.76%），以及G3型阳性率均为100%外，免前其它型中和抗体阳性率和抗体水平均较低，G3型抗体水平差异有统计学意义（P=0.003）。对于免后RV-IgA，在12周龄组中，除G3型RV-IgA的几何平均滴度略低（76:77）外，其它型免后RV-IgA阳转率和抗体水平均较8周龄组高，但差异无统计学意义。提示母传抗体随着时间的推移逐渐衰减，但在我国将疫苗接种时间推迟至12周龄，实际上对消除母传抗体的影响有一定的帮助，但也需要更多年龄组的研究。
在上述接种人群的基础上，将免后任何型RV-IgA阳转的接种者归为血清阳转组，所有型别RV-IgA均未阳转的归为血清未阳转组，分别从两组中随机选取55人和41人通过测序法进行HLA-A、-B、-C、-DRB1、-DQB1分型，计算HLA各位点等位基因型别、单体型和超型在两组人群中的分布频数，统计两组间等位基因、单体型和超型的分布是否有差异，分析HLA基因多态性与轮状病毒减毒活疫苗免疫反应的关系。结果发现HLA-B*40:01在血清未阳转组的分布频率明显高于血清阳转组，差异有统计学意义，与轮状病毒减毒活疫苗接种后无免疫应答相关。
为了评价同时接种OPV的行为是否会影响轮状病毒减毒活疫苗的免疫反应和血清阳转率，本研究以葛兰素史克公司生产的Rotarix作为研究对象，将受试人群随机分为两组。一组按照常规两剂接种程序完成两剂口服Rotarix的接种，并根据国家规划接种年龄接种计划免疫的OPV，但与Rotarix不在同一天接种。另一组在按照常规接种程序完成两剂口服Rotarix的接种基础上，同时接种OPV。分别在首剂接种前、第2剂Rotarix接种后1个月采集血样，进行RV-IgA抗体测定。通过统计分析两组的RV-IgA血清阳转率和水平分布是否有统计学差异。结果显示间隔接种和同时接种组的血清RV-IgA阳转率分别为73.84%和63.95%，差异有统计学意义（P=0.033）；两组的免后RV-IgA抗体几何平均数分别为97和90，差异无统计学意义（P＞0.05）。说明同时接种口服脊髓灰质炎减毒活疫苗可能会影响轮状病毒减毒活疫苗的血清阳转率，但尚未发现其对群体的抗体水平的影响。与同时接种组相比，间隔接种组免后1年血清RV-IgA阳转率和几何平均滴度均较高，差异有统计学意义（P＜0.05）。同时接种组在免后1年中的抗体阳转率和GMT下降更为明显，提示同时接种对RV-IgA抗体水平的维持也有影响。
综上所述，本研究结果表明宿主母传抗体、宿主基因多态性和OPV都可能是影响轮状病毒减毒活疫苗免疫原性的因素，对轮状病毒疫苗的研发、临床试验的设计和免疫程序的制定有一定的参考价值。
 

Rotavirus (RV) is the most common cause of infants’ severe diarrhea around the world. Rotavirus infection is known as a "democratic disease", as it infects children in both developed and developing countries, leading to serious social and disease burden worldwide. But rotavirus infection causes higher under-five mortality rates in developing countries. Vaccination is the only effective way to prevent rotavirus diarrhea and reduce mortality. In recent ten years, the human strain rotavirus vaccine (Rotarix, GlaxoSmithKline, Belgium) and Bovine-human pentavalent reassortant rotavirus vaccine (RotaTeq, Merck＆Co., US) are widely used in the world. Vaccine clinical trials and post market survey data showed that the live attenuated rotavirus vaccines were highly efficacious in developed countries, but not ideal in developing countries where they were most needed. It is urgent to find out the reasons of this phenomenon to improving the effectiveness of rotavirus vaccine in developing countries. A series of studies to explaining this phenomenon have been conducted, and the nutritional status of the vaccinated, intestinal flora, breastfeeding and the prevalence of viral strains were proposed to lead to the variable levels of protection conferred by rotavirus vaccine in different settings. They are not able to give a systemic and reasonable explanation, and the study from other aspects are eagerly needed.
The immune system is a complex network. The factors leading to lower immune response in low-income countries may be not limited to the above. Therefore, in this study, the factors that may impair the immune response of rotavirus attenuated live vaccine were analyzed from the following three aspects: the maternal antibody, the genetic polymorphisms of human leukocyte antigen (HLA) and concomitant administration of oral poliomyelitis vaccine (OPV).
In this study, healthy infants aged 6-13 weeks received three doses of the trivalent reassortant rotavirus vaccine with a interval of one month. Blood samples were collected before the administration of the first dose of vaccine and one month after the third dose of vaccine administration for G2, G3 and G4 types specific RV-IgA. According to whether the G type specific RV-IgA was seroconverted, infants were divided into two groups for each G type respectively. The pre-immunization sera were tested for maternal G2, G3 and G4 specific RV neutralizing antibodies (NT Ab). As a result, the seropositive rate and geometric mean titer (GMT) of pre-administration NT Ab display the following distribution characteristics: G4 type neutralizing antibody was the lowest (49.24%, 12), followed by type G2 (82.95%, 20), and type G3 was the highest (100%, 91). Inversely, the GMT of post-administration G3 type specific RV-IgA was the lowest by 76U/mL, followed by G2 type 86U/mL and G4 type 90U/mL. The positive rate and GMT of any G type pre-administration NT Ab was lower in seroconverters group than that in non-seroconverters group. In addition to the G3 type, the differences of G2 and G4 types NT Ab between the two groups were statistical (P＜0.05), indicating that the maternal antibody may interfere with immune response to live oral rotavirus vaccine.
According to the age of the first dose of vaccine, the subjects were divided into two groups. The mean age was 8 weeks and 12 weeks, respectively. The differences in pre-administration NT Ab and post-administration RV-IgA between the two groups were statistical analyzed to investigate if the delaying schedule can eliminate the influence of maternal antibodies and enhance the immune response of vaccine. As a result, in addition to the seropositive rate of G2 and G3 type NT Ab, vaccinees aged 12 weeks had lower seropositive rate and GMT of pre-immunization NT Ab than infants aged 8 weeks. Inversely, the seroconversion rate and GMT of RV-IgA were higher in the group aged 12 weeks except the GMT of G3 type, but did not show statistical significance. In conclusion, maternal antibodies could gradually decay over time. In our country, it may be helpful in eliminating the influence of maternal antibodies to delay the schedule to 12 weeks of age, but the study on more age group is needed.
On the basis of the above vaccination population, infants with any G type specific RV-IgA seroconverted were classified as seroconverters group. Conversely, infants whose all of G types specific RV-IgA didn’t seroconvert were classified as nonseroconverters group. 55 and 41 subjects belonging to seroconverters and nonseroconverters group respectively were randomly selected to detect the HLA-A, -B, -C, -DRB1 and DQB1 loci genotype by sequence-based typing (SBT) method. The frequencies of each HLA allele, haplotype and supertype were compared between the two groups by statistical analysis to assess the association of polymorphisms of HLA and immune response to rotavirus vaccine. The results showed that the more statistically frequent HLA-B*40:01 was found among seroconverters group infants than nonseroconverters, suggesting that it may be associated with non-response of rotavirus vaccine.
To evaluate if concomitant administration of OPV affect the immune responses and seroconversion to the vaccine, Rotarix was studied as the candidate vaccine. All of the vaccinees received two doses of Rotarix with the normal schedule. According to the administration of OPV, the vaccines were divided into two groups. Concomitant administration was defined as Rotarix and OPV given on the same day; staggered administration as Rotarix and OPV given ≥1 day apart. Blood samples were collected before the administration of the first dose of vaccine，one month and one year after the second dose of vaccine administration for. The differences of RV-IgA seroconversion and GMT between the two groups were analyzed by statistical method. In the respect of RV-IgA in one month after vaccination, infants who received Rotarix and OPV concomitantly were less likely to seroconvert (63.95%) than those who received both vaccines staggered ≥1 day (73.84%, P=0.033). A higher RV-IgA GMT, not achieving statistical significance, in staggered administration group was observed (97:90). Concomitant administration of OPV seems to lower Rotarix seroconversion without influencing the level of antibody in group. About RV-IgA in one year after immunization, infants staggered administrated had significantly higher seroconversion rate and GMT (P＜0.05). The seroconversion rate and GMT decreased more significantly in concomitant administration group, indicating that concomitant administration would affect the maintain of RV-IgA.
    In conclusion，the interrelated factors were illustrated in this study. The maternal antibody, genetic polymorphisms of HLA and concomitant administration of OPV were confirmed to be responsible for the poorer effectiveness and immunogenicity of RV vaccine in developing countries. It has a certain reference value for the research and development of the new rotavirus vaccine in the developing countries, the design of clinical trials and the formulation of the immunization program.