J Virol. 2015; pii: JVI.03566-14.

Regla-Nava JA, Nieto-Torres JL, Jimenez-Guardeño JM, Fernandez-Delgado R, Fett C, Castaño-Rodríguez C, Perlman S, Enjuanes L, DeDiego ML.

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo.

To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy terminal region of E protein led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx and increased CD4⁺ and CD8⁺ T cell counts in the lungs of BALB/c mice, when compared to mice infected with wild type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E* infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels.

These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates.

J Virol. 2015; 89 (2): 970-988.

Perdiguero B, Gómez CE, Cepeda V, Sánchez-Sampedro L, García-Arriaza J, Mejías-Pérez E, Jiménez V, Sánchez C, Sorzano CÓ, Oliveros JC, Delaloye J, Roger T, Calandra T, Asbach B, Wagner R, Kibler KV, Jacobs BL, Pantaleo G, Esteban M.

The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen.

n this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited.

Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors.

Hum Vaccin Immunother. 2014; 10(8).

García-Arriaza J, Esteban M.

Attenuated recombinant poxvirus vectors expressing heterologous antigens from pathogens are currently at various stages in clinical trials with the aim to establish their efficacy. This is because these vectors have shown excellent safety profiles, significant immunogenicity against foreign expressed antigens and are able to induce protective immune responses.

In view of the limited efficacy triggered by some poxvirus strains used in clinical trials (i.e, ALVAC in the RV144 phase III clinical trial for HIV), and of the restrictive replication capacity of the highly attenuated vectors like MVA and NYVAC, there is a consensus that further improvements of these vectors should be pursuit. In this review we considered several strategies that are currently being implemented, as well as new approaches, to improve the immunogenicity of the poxvirus vectors. This includes heterologous prime/boost protocols, use of co-stimulatory molecules, deletion of viral immunomodulatory genes still present in the poxvirus genome, enhancing virus promoter strength, enhancing vector replication capacity, optimizing expression of foreign heterologous sequences, and the combined use of adjuvants. An optimized poxvirus vector triggering long-lasting immunity with a high protective efficacy against a selective disease should be sought.

MBio. 2013 Sep 10;4(5). pii: e00650-13

Almazán F, Dediego ML, Sola I, Zuñiga S, Nieto-Torres JL, Marquez-Jurado S, Andrés G, Enjuanes L.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates.

Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells.

The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection.

J Virol. 2013 Jul;87(13):7282-300

Gómez CE, Perdiguero B, Cepeda MV, Mingorance L, García-Arriaza J, Vandermeeren A, Sorzano CO, Esteban M.

A major goal in the control of hepatitis C infection is the development of a vaccine. Here, we have developed a novel HCV vaccine candidate based on the highly attenuated poxvirus vector MVA (referred as MVA-HCV) expressing the near full-length (7.9 kbp) HCV sequence, with the aim to target almost all of the T and B cell determinants described for HCV.

In infected cells, MVA-HCV produces a polyprotein that is subsequently processed into the structural and nonstructural HCV proteins, triggering the cytoplasmic accumulation of dense membrane aggregates. In both C57BL/6 and transgenic HLA-A2 vaccinated mice, MVA-HCV induced high, broad, polyfunctional and long-lasting HCV-specific T cell immune responses. The vaccine-induced T cell response was mainly mediated by CD8 T cells; however, although lower in magnitude, the CD4+ T cells were highly polyfunctional. In homologous protocol (MVA-HCV/MVA-HCV) the main CD8+ T cell target was p7+NS2, whereas in heterologous combination (DNA-HCV/MVA-HCV) the main target was NS3. Antigenic responses were also detected against other HCV proteins (Core, E1-E2, NS4) but the magnitude of the responses was dependent of the protocol used. The majority of the HCV-induced CD8+ T cells were triple or quadruple-cytokine producers. The MVA-HCV vaccine induced memory CD8+ T cell responses with an effector memory (TEM) phenotype.

Overall, our data showed that MVA-HCV induced broad, highly polyfunctional and durable T cell responses of a magnitude and quality that might be associated with protective immunity and open the path for future considerations of MVA-HCV as prophylactic and/or therapeutic vaccine candidate against HCV.