Construction and Preclinical Evaluation of a Recombinant Attenuated Measles Vaccine Candidate of the H1a Genotype

Abstract

Background: Measles, an acute respiratory infectious disease caused by the measles virus, continues to pose a significant threat to children under five years old worldwide. Despite the availability of effective vaccines, challenges such as insufficient vaccination coverage and antigenic drift contribute to its persistence. Based on a newly isolated wild-type measles virus strain (genotype H1a), designated MVs/Jiangsu.CHN/38.16/1[H1a] (MV-1), this study aims to develop and evaluate a novel recombinant measles virus vaccine candidate designed to enhance immunogenicity and broaden protection against multiple epidemic genotypes. Methods: A recombinant measles virus vaccine candidate, designated rSchwarz/FH(H1a), was developed by incorporating immunogenic genes from the H1a genotype into the backbone of the Schwarz vaccine strain. The genetic stability, safety, and immunogenicity of this vaccine candidate were evaluated in preclinical models. Relevant sample sizes and methodologies were selected to ensure comprehensive assessment of vaccine efficacy against various genotypes (H1a, B3, D8). Results: The rSchwarz/FH(H1a) vaccine candidate demonstrated enhanced immunogenicity, with robust immune responses observed against the targeted genotypes. Additionally, it showed excellent genetic stability and safety profiles, indicating potential for effective use in vaccination programs. Notably, the vaccine provided cross-protection against multiple epidemic genotypes, highlighting its broader application in controlling measles outbreaks. Conclusions: Our findings suggest that the rSchwarz/FH(H1a) vaccine candidate represents a promising advancement in measles vaccine development. It has the potential to strengthen current measles vaccination strategies by providing improved immunogenicity and broader protection against different circulating genotypes. Further clinical trials are warranted to confirm these promising preclinical results.

Keywords: attenuated live; cross-protection; genotype; measles; recombinant virus.

Figure 1

Cross-neutralization analysis of measles virus strains of H1a and A genotype in vivo. Female BALB/c mice as well as female guinea pigs were intraperitoneally injected with Schwarz and MV-1 virus strains at a dose of 1 × 10⁵ CCID₅₀, and second immunization were carried out after 14 days. Each group contained 10 mice or 5 guinea pigs. A total of 14 days after the second immunization, serum samples were collected to determine the neutralizing antibody titers against the Schwarz and MV-1 viruses. The graphs show the geometric mean antibody titers for each group of guinea pigs (A) or mice (B). All animals underwent blood collection and neutralization titer testing prior to immunization. Following a 1:1 (original) dilution of serum from all experimental animals, no viral inhibitory activity was observed in the microneutralization assay, as indicated by 100% cytopathic effect (CPE) while all samples exhibited a titer of <2. Titers of antibody against the Schwarz and MV-1 viruses in sera samples from 18-month-old infants who have received the MMR vaccine were determined by micro-neutralization antibody test (C). All data are expressed in mean with 95% confidence intervals (95% CI). *: 0.01 ≤ p < 0.05; **: 0.001 ≤ p < 0.01; ***: p < 0.001.

Figure 2

Characterization of Measles Virus in which F and H structures were modified. (A) Schematic diagrams show the gene structures of the recombinant measles virus. Gene F or/and H on Schwarz strain were replaced with segments of MV-1 virus to produce recombinant rSchwarz, rSchwarz/F(H1a), rSchwarz/H(H1a), and rSchwarz/FH(H1a). (B) The rSchwarz and rSchwarz/FH(H1a) were observed under electron microscopy. (C) Observation of cytopathic effect (CPE) on measles virus recombinants were performed by infection of Vero and CEF cell lines with gene-modified viruses at a multiplicity of infection (MOI) of 0.01, and cells were imaged under a 10X wide field microscope after four days post-infection. Growth curves of the recombinant viruses on Vero (D) and CEF (E) cells were analyzed by infecting the cells with viruses at 0.01 MOI, and viral titers were determined by using Vero-hSLAM cells incubated with the collected supernatants harvested every 24 h until up to 144 h of infection. (F) In vivo, each of the 10 female BALB/c mice were intraperitoneally injected with rSchwarz, rSchwarz/F(H1a), rSchwarz/H(H1a), and rSchwarz/FH(H1a) at a dose of 1 × 10⁵ CCID₅₀, followed by a second immunization after 14 days. After two weeks of second immunization, mouse sera were collected to measure the neutralizing antibody titers against the Schwarz and MV-1 viruses, respectively. The neutralizing antibody titers were evaluated in serum samples collected from all animals before immunization, and all samples exhibited a titer of <2. Data for viral growth kinetics were expressed as mean ± standard error of the mean (SEM), while neutralizing antibody titers are expressed in mean with 95% confidence intervals (95% CI). **: 0.001 ≤ p < 0.01; ***: p < 0.001.

Figure 3

The amino acid at position 476 of the H protein is a critical determinant for different cross-protection between measles virus strains of genotypes H1a and A. (A) Schematic representation of the construction of recombinant measles virus rSchwarz/H (P397L + N405S), rSchwarzH/(F476L), and rSchwarz/H (P397L + N405S + F476L). This includes detailed amino acid sequences of key antigenic epitopes from both A and H1a genotype measles virus; mutations associated with the MV-1 strain are highlighted in red, while proteins related to the Schwarz strain are indicated in gray. (B) Multistep growth curves of the recombinant viruses in Vero cells. The recombinant viruses were used to infect Vero cells at an MOI of 0.01, and viral supernatants were harvested every 24 h for up to 144 h. Virus titers at each time point were measured in Vero-hSLAM cells. (C) Female BALB/c mice, aged 6–7 weeks and weighing 19–21 g, were intraperitoneally immunized with recombinant measles virus rSchwarz, rSchwarz/H (P397L + N405S), rSchwarzH/(F476L), and rSchwarz/H (P397L + N405S + F476L) at a dose of 5 × 10⁴ CCID₅₀ per animal on days 0 and 14. Each group contained five mice. A total of 14 days after the second immunization, serum samples were collected to determine the neutralizing antibody titers against the Schwarz and MV-1 viruses. Data for viral growth kinetics were expressed as mean ± standard error of the mean (SEM), neutralizing antibody titers are expressed in mean with 95% confidence intervals (95% CI). ns (Not Significant): p ≥ 0.05, *: 0.01 ≤ p < 0.05; **: 0.001 ≤ p < 0.01; ***: p < 0.001. The neutralizing antibody titers were assessed in sera collected from all animals prior to immunization, with all samples showing a titer of <2.

Figure 4

(A) Evaluation of the immunogenicity of recombinant measles virus against multiple genotypes. Each of the 10 female BALB/c mice, aged at 6–7 weeks and weighed between 19 and 21 g, were intraperitoneally immunized with either the rSchwarz or rSchwarz/FH (H1a) viruses at high (1 × 10⁵ CCID₅₀) and low (1 × 10³ CCID₅₀) doses on day 0 and a second immunization on day 14. A total of 14 days after the second immunization, serum samples were collected from the mice. (B) Neutralizing antibody titers of serum samples collected from immunized mice were determined by micro-neutralization antibody test. The graphs show the geometric mean antibody titers for each group of mice. The neutralizing antibody titers were assessed in sera collected from all animals prior to immunization, with all samples showing a titer of <2. All data are expressed in mean with 95% confidence intervals (95% CI). ns (Not Significant): p ≥ 0.05, **: 0.001 ≤ p < 0.01; ***: p < 0.001.

Figure 5

Genetic stability of rSchwarz/FH (H1a) as a vaccine candidate. (A) The rSchwarz/FH (H1a) virus was continuously passaged for 10 passages in primary chicken embryo fibroblast (CEF) cells. Viral samples at passages P0, P7, P9, P12, P17, and P22, were collected, followed by full-genome sequencing using Sanger sequencing. Sequence alignment was performed to analyze positions and details of nucleotide mutations. (B) Growth characteristics of rSchwarz/FH (H1a) virus passaged in CEF cells were confirmed by infecting the CEF cells at 0.01 MOI followed by determination of virus titers by using Vero-hSLAM cells. (C) Each group of five female mice were intraperitoneally immunized with rSchwarz/FH (H1a) viruses from passages P1, P12, and P22 at a dose of 1 × 10⁵ CCID₅₀ on day 0 and 14. A total of 14 days after the second immunization, serum samples were collected from the mice, and titers of neutralizing antibody titers against the Schwarz, D8 and MV-1 measles virus were determined by micro-neutralization antibody test. The neutralizing antibody titers were assessed in sera collected from all animals prior to immunization, with all samples showing a titer of <2. Data for viral growth kinetics were expressed as mean ± standard error of the mean (SEM), while neutralizing antibody titers are expressed in mean with 95% confidence intervals (95% CI).

Figure 6

Safety of rSchwarz/FH (H1a) as a vaccine candidate in Mice. (A) IFNα/βR^−/− (A129) mice (5–6 weeks old) were intracranially administered 10¹⁰ vg rAAV-hsyn-SLAM, followed 60 days later by a single intracranial challenge with 30 μL of measles virus (3 × 10⁴ CCID₅₀). Each group contained four animals. Body weight was monitored for 14 days post-challenge (D0), and mortality was observed for 22 days. (B) Statistical analysis of mouse body weight changes 14 days after challenge in the DMEM, MV-1, rSchwarz, and rSchwarz/FH(H1a) groups. (C) Mortality statistics 22 days after challenge in the DMEM, MV-1, rSchwarz, and rSchwarz/FH(H1a) groups.

Figure 7

Safety of rSchwarz/FH (H1a) as a vaccine candidate in Rhesus Macaques. (A) Measles antibody-negative rhesus macaques were divided into three groups by body weight: DMEM control (n = 4), S191 (n = 10), and rSchwarz/FH(H1a) (n = 10). Each received a single bilateral intrathalamic injection of 1 mL solution (10^4.25 CCID₅₀ measles virus or DMEM). Clinical signs and body weights were monitored for 31 days. Serum samples for neutralizing antibody titers against Schwarz, MV-1, and D8 strains were collected on day 21. Animals were euthanized on day 31 for histopathological examination of brain and spinal cord. (B) Neutralizing antibody titers against the Schwarz, MV-1, and D8 measles virus were measured by micro-neutralization antibody test in serum samples collected from the DMEM, S191, and rSchwarz/FH(H1a) groups on day 21 post-injection. The graphs show the geometric mean antibody titers for each group of rhesus macaques. The neutralizing antibody titers were assessed in sera collected from all rhesus macaques prior to immunization, with all samples showing a titer of <2. And the neutralizing titers of the immune sera from the DMEM group were also all <2. Data for longitudinal changes in body weight and temperature of nonhuman primates (NHPs) were expressed as mean ± standard error of the mean (SEM). Neutralizing antibody titers were presented as mean with 95% confidence intervals (95% CI). **: 0.001 ≤ p < 0.01; ***: p < 0.001. (C) Statistical analysis of seroconversion rates, a neutralizing titer of >2 was considered positive. (D) Post-injection body weight changes in DMEM (monitored until day 31), S191, and rSchwarz/FH(H1a) (both monitored until day 21) groups compared. (E) Post-injection body temperature changes in DMEM (monitored until day 31), S191, and rSchwarz/FH(H1a) (both monitored until day 21) groups compared. (F) H&E-stained histological sections of brain and spinal cord tissues from monkeys injected with DMEM, S191, or rSchwarz/FH(H1a), examined at 100× magnification.