Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350

Abstract

Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1(st) or the 3(rd) position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3(rd) position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation.

Keywords: Cotton rats; EBV gp350; Edmonston-Zagreb; Measles vaccine; RSV F; Rhesus monkeys..

Fig. (1)

Construction of measles vaccine vectors encoding ecto domains of RSV F and EBV gp350 and protein expression in virus-infected Vero cells. (A) RSV F ectodomain and EBV gp350 ectodomains were inserted in the 1^st and 3^rd transcription units of the measles Zagreb strain genome. Insertion was performed in the non-coding region 3' proximally of the MV N gene or between P and M genes of pEZ under the control of additional measles transcriptional units containing gene stop (GE)-gene start (GS) sequences. *RSV F gene (aa. 1-524) was inserted into EcoRV site using blunt ligation. ^#EBV gp350 full length (aa. 1-960), EBV gp350 truncated (aa. 1-470) or RSV F gene was inserted between EcoRV and AfeI site. (B) Vero cells were infected at an MOI of 3.0 with measles viruses encoding soluble RSV F (sF1 or sF3) or soluble EBV gp350 (gp350 or gp350 tr). After 18 h, western blots were used to detect presence of measles virus nucleoprotein in infected Vero cell lysates or RSV F protein, EBV gp350 protein in the supernatant. Black arrowheads indicate the predicted molecular masses of the proteins. Relative intensity of the MV N protein bands was expressed as the intensity of the band relative to the rEZ control.

Fig. (2).

Growth kinetics of measles vectors encoding soluble RSV F or soluble EBV gp350 was similar to that of recombinant measles constructs. Vero cells were infected at MOI of 0.01 with measles vectors. Cells were incubated at 35°C for 7 days. Cells and supernatant were sampled daily for virus titers using standard plaque assay. * denotes statistically significant difference in titer of sF3 versus sF1, rEZ and EZ using the Kruskal-Wallis test.

Fig. (3).

RSV F-specific and EBV gp350-specific splenocytes were induced in the spleens of cotton rats immunized i.m. with measles vectors. Cotton rats were vaccinated on days 0 and 28 with measles vectors. Splenocytes were harvested 7 days after the second measles vectors vaccination. A total of 3 x 10⁵ cells were stimulated with either RSV F protein (A and C) or EBV gp350 protein (B and D) in vitro for 20 h, and the numbers of IFN-γ spot-forming cells (A and B) and IL-4 spot-forming cells (C and D) were quantified by an ELISPOT assay. Spots were counted with an automated counting device and are expressed as numbers of spots per 10⁶ cells. Groups are compared using Mann-Whitney rank sum test. Each symbol represents an individual animal.

Fig. (4).

Reduction of titers of RSV in the lungs of cotton rats vaccinated with measles-vectored RSV F after challenge. Naive cotton rats were immunized with 10⁵ PFU of sF1 or sF3 on day 0 and 28. Cotton rats were then challenged with 10⁶ PFU of RSV A2 on day 56. Lungs were harvested on day 60 and RSV titers were determined in the lung homogenates by standard plaque assay. Each experimental group represents 5 individual animals except in UVinactivated sF3 group (n=3).

Fig. (5)

RSV F specific antibody response and cellular response in rhesus macques vaccinated with measles vectors. Measles seronegative and sero-positive rhesus macaques were immunized with 10⁵ PFU of sF3 or gp350 on day 0 and 28. (A) RSV F titers were measured on day 28 (post dose 1) and 42 (post dose 2) using ELISA. (B) PBMCs were isolated from whole blood on day 56. The numbers of IFN-γ spot forming cells were quantified with an ELISpot assay when PBMCs were stimulated with measles viral antigens, RSV F or EBV gp350 antigens in vitro. Spots were counted with an automated counting device and are expressed as numbers of spots per 10⁶ cells ± SEM. ND = not determined.