Attenuation and immunogenicity of host-range extended modified vaccinia virus Ankara recombinants

Abstract

Modified vaccinia virus Ankara (MVA) is being widely investigated as a safe smallpox vaccine and as an expression vector to produce vaccines against other infectious diseases and cancer. MVA was isolated following more than 500 passages in chick embryo fibroblasts and suffered several major deletions and numerous small mutations resulting in replication defects in human and most other mammalian cells as well as severe attenuation of pathogenicity. Due to the host range restriction, primary chick embryo fibroblasts are routinely used for production of MVA-based vaccines. While a replication defect undoubtedly contributes to safety of MVA, it is worth considering whether host range and attenuation are partially separable properties. Marker rescue transfection experiments resulted in the creation of recombinant MVAs with extended mammalian cell host range. Here, we characterize two host-range extended rMVAs and show that they (i) have acquired the ability to stably replicate in Vero cells, which are frequently used as a cell substrate for vaccine manufacture, (ii) are severely attenuated in immunocompetent and immunodeficient mouse strains following intranasal infection, (iii) are more pathogenic than MVA but less pathogenic than the ACAM2000 vaccine strain at high intracranial doses, (iv) do not form lesions upon tail scratch in mice in contrast to ACAM2000 and (v) induce protective humoral and cell-mediated immune responses similar to MVA. The extended host range of rMVAs may be useful for vaccine production.

Keywords: Attenuated live vaccines; Recombinant vaccinia virus; Virus pathogenesis; Virus vectors.

Fig. 1

Replication of rMVAs. (A) Cell-to-cell virus spread. Monolayer of BS-C-1 and Vero cells were infected with MVA, rMVA 44/47.1 and rMVA 51.1, Ankara and ACAM2000 viruses. At 48 h after infection, the cells were fixed and immunostained with broadly reactive anti-VACV antibody followed by horseradish peroxidase conjugated to anti-rabbit immunoglobulin. Arrows point to the pin-point foci formed by MVA. (B) Growth curve. Vero cells were infected with 0.1 pfu/cell of MVA, rMVA 44/47.1, rMVA 51.1 or ACAM2000 and harvested at the indicated times post infection. Cell-associated viruses were titrated on CEF monolayers in the case of MVA and rMVAs and BS-C-1 cells for ACAM2000. Standard error bars shown. (C) The ratios of virus output to virus input are plotted with standard error bars.

Fig. 2

Comparison of virulence in the intranasal challenge model. Groups (n=5) of 5- to 6-weeks-old female mice (BALB/c C57BL/6 and ICR-SCID) were infected intranasally with 10⁶ PFU of MVA, rMVA 44/47.1 and rMVA 51.1, Ankara and ACAM2000 viruses. The uninfected group was mock infected with diluents. Animals were monitored daily for weight loss and death for 47 days although only 14 are shown in the figure. Error bars indicate standard error. Asterisks in BALB/c and 57BL/6 groups indicate numbers of mice infected with Ankara that were sacrificed due to weight loss >30%. In ICR-SCID mice group infected with Ankara, the asterisks refer to two deaths and three sacrificed mice. One ACAM2000-infected SCID mouse was sacrificed due to weight loss >30% at day 47. The postmortem was positive for ACAM2000 virus.

Fig. 3

Comparison of virulence in intracranial challenge model. (A) Five- to six-weeks-old female BALB/c mice were infected intracranially with 10⁶ PFU of MVA (n=10), rMVA 44/47.1 (n=15) and rMVA 51.1 (n=15) and ACAM2000 (n=15) viruses. The weight control group was mock infected with diluents. Animals were monitored daily for weight loss for 21 days with no deaths. The data shown are combined from two independent experiments. Error bars indicate standard error. (B) Experimental details are the same as in panel A except that the mice were infected with 10⁷ pfu of MVA (n=15), rMVA 44/47.1 (n=15) and rMVA 51.1 (n=15) and ACAM2000 (n=15) viruses. (C) All 15 mice infected with ACAM2000, 14 mice infected with rMVA 44/47.1, 10 mice infected with rMVA 51.1 and 1 mouse infected with MVA were sacrificed on days 4 – 5 due to >30% loss of starting weight.

Fig. 4

Lesion formation following tail scarification. Groups (n=3) of 5-weeks-old BALB/c mice were inoculated by tail scratch with 10⁸ PFU of MVA, rMVA 44/47.1 or rMVA 51.1 or with 2×10⁵ pfu of ACAM2000 or PBS containing 2% fetal bovine serum. Mice were inspected for lesion formation. Images were made on day 12.

Fig. 5

Vaccine-induced immune responses. (A) IgG antibodies. Groups of mice (n=4) were vaccinated intramuscularly or by tail scratch with 10⁵, 10⁶, 10⁷ and 10⁸ pfu of MVA, rMVA 44/47.1 or rMVA 51.1 intramuscularly or by tail scarification. Four mice were inoculated by tail scratch (t.s.) with 2×10⁵ pfu of ACAM2000. The intramuscular vaccination with 10⁵ pfu was repeated with 10 mice for each virus strain and the data combined with that of the initial experiment. Animals were bled 3 weeks post vaccination. Serum ELISA IgG titers were determined using 96-well plates coated with purified MVA. Geometric mean titers (GMT) were determined. The error bars represent geometric standard deviations. In some cases the values were so close that the error bars were not resolved. (B) CD8+ T cells. Mice (n=5) were inoculated intramuscularly (i.m) with 10⁶ pfu of MVA, rMVA 44/47.1 or rMVA 51.1 or by tail scratch (t.s.) with 2 ×10⁵ pfu of ACAM2000. After seven days, splenocytes were processed as described in Materials and methods and the percentage of CD8⁺ splenocytes that were positive for IFNγ, TNFα, and IL-2⁺ was determined by flow cytometry.

Fig. 6

Protection of BALB/c mice against lethal VACV WR challenge. (A) Groups (n=4) of 6-week-old female BALB/c mice were vaccinated intramuscularly (i.m.) or by tail scrach (t.s.) with MVA, rMVA 44/47.1 and rMVA 51.1 at 10⁵ to 10⁷ pfu or with 2×10⁵ pfu of ACAM2000 by tail scratch. Challenge control group (n=4) was mock vaccinated intramuscularly with diluents. Four weeks later, the animals were challenged intranasally with 10⁷ pfu of VACV WR, and monitored for 21 days for weight loss and death. All of the challenged mice in the control group died. Asterisks in 10⁵ pfu t.s. groups represent individual mice that were sacrificed. The asterisk in 10⁶ pfu t.s. group on day 7 represented a death and on day 8 a sacrifice (B) Groups (n=10) of mice were vaccinated intramuscularly with 10⁵ pfu of MVA, rMVA 44/47.1 or rMVA 51.1 or with 2.5 × 10⁵ pfu of ACAM2000 and challenged with VACV WR as described in panel A. The challenged controls were found dead, whereas asterisks in the vaccinated groups indicate sacrifice due to loss of 30% of starting weight.