The attenuated NYCBH vaccinia virus deleted for the immune evasion gene, E3L, completely protects mice against heterologous challenge with ectromelia virus

Abstract

The New York City Board of Health (NYCBH) vaccinia virus (VACV) vaccine strain was deleted for the immune evasion gene, E3L, and tested for its pathogenicity and ability to protect mice from heterologous challenge with ectromelia virus (ECTV). NYCBHΔE3L was found to be highly attenuated for pathogenicity in a newborn mouse model and showed a similar attenuated phenotype as the NYVAC strain of vaccinia virus. Scarification with one or two doses of the attenuated NYCBHΔE3L was able to protect mice equally as well as NYCBH from death, weight loss, and viral spread to visceral organs. A single dose of NYCBHΔE3L resulted in low poxvirus-specific antibodies, and a second dose increased levels of poxvirus-specific antibodies to a level similar to that seen in animals vaccinated with a single dose of NYCBH. However, similar neutralizing antibody titers were observed following one or two doses of NYCBHΔE3L or NYCBH. Thus, NYCBHΔE3L shows potential as a candidate for a safer human smallpox vaccine since it protects mice from challenge with a heterologous poxvirus.

Identification of protective T-cell antigens for smallpox vaccines

“…Nevertheless, VV-NPVTVINEY-specific CD8+ T-cells from donors 1 and 2 recognized the variola epitope in IFN-λ ELISpot assays ( Figure 4A). At an E:T ratio of 40:1, donor 1 T cells killed NPV-pulsed target cells (89.9% 51 Cr release), NPV V!I pulsed target cells (100% 51 Cr release) and control target cells (10.1% 51 Cr release), while donor 2 T-cells induced 59.2%, 59.2% and 2.1% killing of the same target cells, respectively ( Figure 4B). To validate this cross-reactivity, we generated an HLA-B35/NPV V!I (variola) tetramer that recognized 7.6% and 0.5% of the E3L stimulated CD8 T-cells from donors 1 and 2.…”

“…The cytotoxic activity of E3L-specific T cells was tested in a standard 6-h 51 Cr release assay using autologous or allogeneic (HLA-mismatched or matched at a single allele) OKT3 blasts. OKT3 blasts were pulsed with E3L pepmixes (50 ng) at 37°C for 30 min.…”

“…OKT3 blasts were pulsed with E3L pepmixes (50 ng) at 37°C for 30 min. Target cells were labeled with 51 Cr sodium chromate (MP Biomedicals, Solon, OH, USA) at 37°C for 1 h, then washed three times and seeded in triplicate in 96-well V-bottomed plates (Falcon) at 5 £ 10 3 cells per well. Effector cells were added at 40:1, 20:1, 10:1 and 5:1 effector:target (E:T) ratio.…”

“…To validate the response to peptide 22 we tested the ability of donor 1 E3L-specific CD8+ T cells to kill autologous activated T cells alone (negative control) or pulsed with peptide 22, in a 6 h 51 Cr release assay ( Figure 2D). At an effector:target (E:T) ratio of 20:1, T cells killed peptide 22-pulsed target cells (45.5% specific 51 Cr release), with minimal recognition of control target cells (2% specific 51 Cr release). Cytotoxicity was blocked by Class I MAbs (14.6% specific killing) ( Figure 2D).…”

Modulating Vaccinia Virus Immunomodulators to Improve Immunological Memory

“…NYCBHΔE3 vaccination by scarification was protective in alternative models as well, i.e., rabbitpox virus (RPXV) infection in rabbits, ECTV in mice and MPXV in cynomolgus macaques. Whilst in mice, vaccination with one or two doses of NYCBHΔE3 protected against a ECTV challenge, in rabbits, two doses of NYCBHΔE3 were necessary to protect against high-dose RPXV challenge in comparison to a single dose of NYCBH [ 130 , 131 ]. In rabbits, vaccination with NYCBHΔE3 did not prevent development of secondary lesions in RPXV-challenged animals, though two doses of NYCBHΔE3 were as protective as one dose of NYCBH and both strains induced comparable titres of anti-VACV neutralising antibodies [ 130 ].…”