严重急性呼吸综合征冠状病毒2（Severe Acute Respiratory Syndrome Coronavirus-2，SARS-CoV-2）在传播过程中不断演化出新的变异株。这些变异株通过关键抗原表位位点的突变，实现对既往感染或疫苗接种建立的免疫屏障的逃逸，从而导致人群中突破性感染的出现。因此，亟需开发具有广泛保护作用的疫苗以应对变异株的出现。

在本研究中，首先通过抗原表位预测的方法，寻找对蛋白抗原表位影响较大且有助于免疫逃逸的热点突变，并将其整合到受体结合域（Receptor binding domain，RBD）蛋白中，从而获得合成RBD（Complex RBD，cRBD）蛋白。本研究共设计了三条cRBD蛋白序列，分别为cRBD1、cRBD2、cRBD3。随后利用大肠杆菌表达系统以及Ni²⁺亲和层析对蛋白进行表达纯化，与佐剂混合后对小鼠进行免疫。实验结果表明，免疫后的小鼠血清中炎症相关细胞因子水平在短时间内升高，说明产生了免疫应答；免疫14天后检测到高水平的RBD特异性IgG结合抗体，表明疫苗具有较好的免疫原性。为进一步探究其体液免疫效果与中和抗体（Neutralizing Antibody，nAb）交叉保护的广谱性，我们检测了免疫后小鼠血清对多个SARS-CoV-2变异株的nAbs水平，发现其对所有检测的变异株均具有较高的中和活性，其中包括原始株、Alpha、Beta、Delta、BA.1、BA.2、BA.5、XBB.1.5、XBB.1.16、EG.5.1、EG.5.1.1、BF.7、BQ.1.1、CH.1.1、BA.2.86及JN.1。结果提示，cRBDs疫苗诱导产生的nAbs对绝大部分SARS-CoV-2变异株具交叉保护作用，其具备成为广谱性疫苗的潜力。随后进一步了运用ELISPOTS方法检测了cRBDs疫苗的细胞免疫水平，使用BA.1.1，XBB及EG.5的S蛋白作为刺激物，刺激脾脏淋巴细胞后检测IFN-γ、IL-2、IL-4的分泌情况，结果表明三者均能够刺激各免疫组细胞产生IFN-γ、IL-2、IL-4细胞因子，其中cRBD3组刺激后产生的IFN-γ相对最多，说明cRBDs疫苗具有较好的细胞免疫效果。最后，选择部分代表性的变异株对cRBDs疫苗免疫后的小鼠进行攻毒保护实验，结果表明cRBDs对检测的各变异株均具有保护效果，尤其在减轻感染引起的肺部损伤方面表现显著。在各项检测中，基于cRBD3的疫苗表现出了最为广泛的保护效力。为了探寻cRBDs广谱性的机制，对免疫后小鼠的全血进行B细胞受体（B cell receptor，BCR）测序，发现使用特定V-J对的免疫球蛋白在cRBD3免疫后小鼠中被显著诱导表达，推测其可能是cRBD3广谱性实现的原因。

综上所述，本研究证实了将导致免疫逃逸的热点突变纳入RBD以扩大抗原诱导的抗体活性谱的可行性，为广谱新型冠状病毒疫苗的研发提供了新思路。

New variants of SARS-CoV-2 have continuously evolved during transmission, potentially overcoming the immune barriers established by previous infections or vaccinations through mutations at key antigenic epitope sites, thereby leading to the emergence of breakthrough infections within the population. Therefore, there is an urgent need to develop vaccines with broad protective efficacy against the emergence of variants.

In this study, cRBDs protein were designed by integrating the hot spots mutations that have great impact on antigenic epitopes and contribute to immune evasion into the original RBD protein. In this study, a total of three cRBD protein sequences were designed, namely cRBD1, cRBD2, and cRBD3. The protein was then expressed using the E.coli expression system and subsequently purified using Ni-affinity chromatography followed by immunization in mice after mixing with an adjuvant. The experimental results showed that the level of inflammation-related cytokines in the serum of immunized mice increased shortly after, indicating the immune response; high levels of RBD-specific IgG were detected 14 days after immunization, suggesting good immunogenicity of the vaccine. To further explore its humoral immune effects and the broad-spectrum protective potential of neutralizing antibodies, we measured the levels of nAbs in the serum of immunized mice against multiple SARS-CoV-2 variants and found high neutralizing activity against all tested variants, including the original strain, Alpha, Beta, Delta, BA.1, BA.2, BA.5, XBB.1.5, XBB.1.16, EG.5.1, EG.5.1.1, BF.7, BQ.1.1, CH.1.1, BA.2.86, and JN.1. The results suggest that the nAbs by induced cRBDs vaccine exhibit cross-protective effects against most SARS-CoV-2 variants, demonstrating potential as a broad-spectrum vaccine. Furthermore, we employed the Elispot method to assess the cellular immune response of the cRBDs vaccine, using the S proteins of BA.1.1, XBB, and EG.5 as stimulants to detect the secretion of IFN-γ, IL-2, and IL-4 after stimulating splenic lymphocytes. The results indicated that all three immunized groups could produce IFN-γ, IL-2, and IL-4 cytokines after stimulated, with the cRBD3 group showing the highest relative production of IFN-γ post-stimulation, indicating a robust cellular immune response of the cRBDs vaccine. Finally, we conducted challenge protection experiments in mice immunized with the cRBDs vaccine using representative variants, and the results showed that the cRBDs vaccine provided protection against all tested variants, particularly in reducing lung damage caused by infection. The cRBD3-based vaccine demonstrated the most extensive protective efficacy in all tests. To explore the mechanism of the broad-spectrum nature of cRBDs, BCR sequencing was performed on the whole blood of immunized mice, revealing that immunoglobulins using specific V-J pairs were significantly induced in mice post cRBD3 immunization, which may account for the broad-spectrum effectiveness of cRBD3.

In summary, this study confirms the feasibility of incorporating hotspot mutations that lead to immune evasion into the RBD to expand the spectrum of antibody activity induced by antigens. It provides a new perspective for the development of broad-spectrum vaccines against the novel coronavirus.