Attenuated and replication-competent vaccinia virus strains M65 and M101 with distinct biology and immunogenicity as potential vaccine candidates against pathogens

Abstract

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4(+) and CD8(+) T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4(+) whereas DNA-LACK/M101-LACK preferentially induced CD8(+) T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.

Fig 1

Viral growth efficiency and plaque size phenotype in cultured cells. (A) CEF, 3T3, and HeLa cells were infected at an MOI of 0.01 PFU/cell, collected at 6, 24, and 48 h.p.i., frozen and thawed three times, and sonicated, and virus infectivity was titrated by a plaque assay in BSC-40 cells. (B) Comparison of virus yields in CEF cells infected at an MOI of 0.01 PFU/cell and an MOI of 5 PFU/cell. Data are represented as means ± standard deviations (SD). (C) Plaque size in BSC-40 cells at 48 h.p.i. after immunostaining, comparing the WR and MVA strains versus M65 and M101 mutants. Magnification, ×10.

Fig 2

Kinetics of luciferase expression and viral production in mice. BALB/c mice (3 per group) were inoculated i.p. with 2 × 10⁷ PFU/mouse of WR-Luc, M65-Luc, M101-Luc, or MVA-Luc. Tissues were collected at 6, 24, 48, and 72 h.p.i., and luciferase expression and viral titers were determined as described in Materials and Methods. Results represent mean values of luciferase from samples of three animals per time expressed as RLU per mg of protein (left panels) or viral production expressed as PFU per mg of protein (right panels). Data are representative of the results of three different experiments.

Fig 3

Weight loss of mice inoculated with different viruses. (A) Immunization schedule. BALB/c mice (6 to 8 weeks old; 3 per group) received 10⁷ or 10⁸ PFU of M65-wt or M101-wt virus or 10⁵ PFU of WR virus by the intranasal (i.n.) route. At 18 days after the first inoculation, 10⁷ PFU, equivalent to 100 lethal doses (LD) of WR virus, was delivered i.n. to all the groups. (B) Monitoring of BALB/c mouse weight loss with time. Asterisks show P values of differences between M65 and M101 at the same dose. (C) Immune system-suppressed mouse schedule. C57BL6 mice (12 weeks old; 4 per group) received 300 mg/kg of cyclophosphamide by the i.p. route 5 days before the immunization and a second dose of 150 mg/kg 24 h before the inoculation of the different viruses. Animals treated with cyclophosphamide or mock treated with PBS received 10⁸ PFU of M65 or M101 virus or 10⁵ of WR virus by the i.n. route. (D) Monitoring of C57BL6 mouse weight loss with time. Symbols are given for each virus.

Fig 4

Histopathological studies in lungs of animals infected with mutant viruses M65 and M101. At day 5 postinfection of mice inoculated as described for Fig. 3A, one animal of each group was sacrificed and lungs were extracted for histological studies (magnification, ×10). (A) Hematoxilin-eosin staining was performed in order to identify signs of tissue infection such as leukocyte extravasations and accumulation, bronchiole epithelium modification, and presence of interstitial hemorrhage. Uninfected and doses of virus infection are indicated. (B) The presence of VACV proteins in the lung was analyzed by immunohistochemistry using rabbit polyclonal antibodies against VACV (panels indicated as RαWR). The brown color denotes virus-specific antigens. Sections incubated with secondary antibody were used as control staining (panels indicated as “Control”).

Fig 5

Diagrams of the genomes of M65 and M101 viruses. Genes deleted or mutated in both genomes are shown in boxes. In the scheme, the deletion in the left end of the genome in both viruses and the small deletion in the right end of M101 virus (both deletions shown in red) can be observed. Different colors are used to denote gene functions.

Fig 6

Schematic representation of the deletions present in the left end (A) or right end (B) of the genome of different strains of vaccinia virus. The gene nomenclature corresponds to the Copenhagen genome wherever possible. Numbers represent the names of the genes in WR nomenclature. Numbers with asterisks are in MVA nomenclature. Designations of truncated genes are underlined. Truncated genes are represented by reduced-size empty arrows.

Fig 7

Evaluation of protection conferred by MVA-LACK, M65-LACK, and M101-LACK recombinant viruses in heterologous prime/boost vaccination regimen. BALB/c mice (6 per group), 6 to 8 weeks of age, were primed intradermally (i.d.) in the abdomen with 100 μg of DNA-LACK or DNA-φ in a 100-μl volume per mouse. At day 14, mice were boosted intraperitoneally (i.p.) with 2 × 10⁷ PFU/mouse of M65-LACK, M101-LACK, MVA-LACK, nonrecombinant parental virus, or PBS/PBS. Graphs represent development of lesions after different challenges with leishmania parasites: 5 × 10⁴ L. major metacyclic promastigotes 3 weeks after the booster (A), 5 × 10⁴ L. major metacyclic promastigotes 8 weeks after the booster (B), and 2 × 10⁴ L. amazonensis stationary-phase synchronized promastigotes 8 weeks after the booster (C). Asterisks show P values for the different groups. Right panels represent each mouse lesion size measurement (closed symbol) at the experiment endpoint. The SD and mean values are indicated by vertical and horizontal bars, respectively.

Fig 8

Memory immune response in mice immunized with the different mutant viruses. BALB/c mice (4 per group) were immunized as described for Fig. 7. (A) Analysis of the phenotype of memory antigen-specific CD4⁺ and CD8⁺ T cells in splenocytes restimulated with A20 cells nucleofected with pCINeo-LACK. Memory T cells were classified as central memory (CD62L⁺ CD127⁺), effector memory (CD62L⁻ CD127⁺), or effector (CD62L⁻ CD127⁻). Percentages represent the frequencies of T cells secreting IFN-γ and/or TNF-α and/or IL-2. (B to D) Within the lymphocyte population, T cells were gated and analyzed for IFN-γ, TNF-α, and/or IL-2 production. Cytokine production by LACK-specific CD8⁺ T cells (B), LACK-specific CD4⁺ T cells (C), and LACK157-173 peptide-specific CD4⁺ T cells (D) was analyzed. The different combinations of cytokines are indicated on the x axis; percentages of T cells producing any cytokine are indicated on the y axis. The different pies show the quality of the response measured as the relative quantities of single-, double-, or triple-cytokine-producing cells. (E) Humoral response to the viral vector, measured by ELISA in serum (1:50 dilution) of infected mice at 11 days (adaptive) or 8 weeks (memory) post-booster immunization. O.D, optical density.