• Vaccine Ontology ID: VO_0004092
• Type: Replication-defective virus
• Preparation: A vial of MVA passage 572 was plaque-purified, propagated in chick embryo fibroblasts, and purified by sedimentation through a sucrose cushion (Earl et al., 2004).
• Virulence: (Earl et al., 2004; Meseda et al., 2005)
• Description: Modified vaccinia Ankara (MVA) has been studied most extensively out of the replication-defective vaccines. MVA has an excellent safety profile and could be used in groups in whom Dryvax is currently contraindicated. MVA was given to 120,000 people in Germany in the 1970s, followed by vaccination with live virus Elstree. MVA was safe but was not field-tested because smallpox was not present in Europe at that time. MVA has since been evaluated in animal models and in human studies. In phase I human clinical trials, MVA was found to be safe and immunogenic on its own and found to prime for greater immune responses and attenuate lesion formation if given in advance of Dryvax vaccination. MVA is also being evaluated in persons with contraindications to live virus vaccine such as atopic dermatitis and immunosuppression (Parrino et al., 2006).

• Vaccine Ontology ID: VO_0004097
• Type: Highly attenuated clone
• Preparation: MVA-BN has been derived via additional passages in serum free chicken embryo fibroblast (CEF) cultures, and is replication incompetent in mammalian cell lines, avirulent even in immune compromised hosts, highly immunogenic in mammalian animal models, and may be administered both s.c. and i.m. The vaccine was produced by IDT under Good Manufacturing Practice (GMP) conditions and provided by Bavarian Nordic as a liquid frozen product stored at −80 °C. Doses of 2 × 10^6, 2 × 10^7, 2 × 10^8 TCID50/ml were formulated in 10 mM Tris, 140 mM NaCl, pH 7.4. The vaccine was thawed and 0.5 ml were administered to subjects to deliver a dose of 10^6, 10^7, 10^8 TCID50, respectively (Vollmar et al., 2006).
• Virulence: (Vollmar et al., 2006)
• Description: MVA-BN (IMVAMUNE) was developed from the Modified Vaccinia Ankara strain (MVA) that was used as a priming vaccine prior to administration of conventional smallpox vaccine in a two-step program and shown to be safe in more than 120,000 primary vaccinees in Germany and used as a veterinary vaccine to protect against several veterinary orthopoxvirus infections (Vollmar et al., 2006).

• Vaccine Ontology ID: VO_0004093
• Type: Replication defective virus
• Preparation: NYVAC was derived from the Copenhagen strain and developed by selective deletion of 18 open reading frames (ORFs) (Parrino et al., 2006).
• Virulence: (Belyakov et al., 2003; Edghill-Smith et al., 2003)
• Description: Smallpox vaccination induced significantly larger skin lesions in immunocompromised macaques than in healthy macaques. Vaccination of immunocompromised macaques with the genetically-engineered, replication-deficient poxvirus NYVAC, before or after retrovirus infection, was safe and lessened the severity of Dryvax-induced skin lesions. Neutralizing antibodies to vaccinia were induced by NYVAC, even in macaques with severe CD4+ T cell depletion, and their titers inversely correlated with the time to complete resolution of the skin lesions. Together, these results provide the proof of concept, in macaque models that mirror human immunodeficiency virus (HIV) type 1 infection, that a prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination, and they highlight the importance of neutralizing antibodies in protection against virulent poxvirus (Edghill-Smith et al., 2003).

Human Response

• Host Strain: Adult males
• Vaccination Protocol: Healthy male subjects aged 20–55 years were eligible for recruitment. The study design was divided into two parts: Part I subjects (n = 68) had no prior history of smallpox vaccination, while Part II (n = 18) subjects had a prior history of smallpox vaccination, documented by a vaccination certificate or a typical vaccination scar. Part I subjects were randomly assigned to receive a dose of either 10^6 (Group 1, n = 18), 10^7 (Group 2, n = 16), 10^8 (Group 3, n = 16) TCID50 MVA-BN in a double-blind manner, or 10^8 TCID50 (Group 4, n = 18) open-label, on day 0 and d28. Part II participants received a single dose of 10^8 TCID50 (Group 5, n = 18), open-label, to evaluate a boost response in previously vaccinated subjects. Study-specific assessments were conducted at screening and on d 0, 7, 28, 35, 42, and 126 (Vollmar et al., 2006).
• Persistence: T-cell immunity can persist for up to 50 years after immunization (Vollmar et al., 2006).
• Side Effects: 15 of the 64 general adverse events were assessed as possibly related to the study vaccine. 2 of these each occurred in Groups 1, 3, and 4, respectively, and 9 (28%) in the pre-immunized subjects. Only 1 serious adverse event was reported during the study: a subject in Group 4 was hospitalized due to an infected epidermal cyst on the face, 12 days after the second injection; however, this event was judged to be unrelated to the study vaccine, and the subject recovered without sequelae (Vollmar et al., 2006).
• Efficacy: The immune responses achieved after administration of MVA-BN were highly dose-dependent. Total IgG seropositivity rates, as determined by the ELISA, reached a maximum of 81% and 88% following a single vaccination using the highest dose (10^8 TCID50) via the s.c. or i.m. routes, respectively. They reached 100% following the second vaccination. On the other hand, the pre-immunized subjects attained 100% seropositivity after a single vaccination with MVA-BN although only 4 of these subjects had detectable antibody titers prior to inclusion, implying a pre-existing boostable immunity more than 20 years post-vaccination (Vollmar et al., 2006).
• Description: The primary objective of the study was to demonstrate safety and tolerability of MVA-BN at different doses administered to healthy subjects with or without a history of smallpox vaccination. Immunogenicity was assessed in all subjects as a secondary endpoint and was also used to evaluate dose-related responses and optimal route of application (Vollmar et al., 2006).

Mouse Response

• Host Strain: BALB/cByJ
• Vaccination Protocol: To compare the effectiveness of various routes of MVA immunization, male BALB/cByJ mice (obtained from the Jackson Laboratory, Bar Arbor, ME) were immunized through 3 different routes at doses from 10^6 to 10^8 pfu, and sera were collected every 3 weeks for 15 weeks for evaluation of Dryvax-specific antibody by ELISA using inactivated virus (Meseda et al., 2005).
• Persistence: The antibody response to vaccination was observed in mice over a relatively long period of time (12–15 weeks) following the initial dose of vaccine. By each measurement, the elicited immune response was stable over this time frame for both Dryvax and MVA. Further, when animals received a second dose of MVA, the antibody response was elevated compared to a single immunization, and was stable for the remainder of the observation period (6 to 9 weeks) (Meseda et al., 2005).
• Side Effects: A safer smallpox vaccine could benefit the millions of people that are advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. Because the correlates of smallpox protection are unknown, findings of similar humoral and cellular immune responses to MVA and Dryvax in NHPs and substantial protection against a severe monkeypox virus challenge are important steps in the evaluation of MVA as a replacement vaccine for those with increased risk of severe side effects from the standard live vaccine, or as a pre-vaccine. As a result of extreme attenuation, MVA causes no adverse effects even when high doses are injected into immunedeficient NHPs. No adverse local or systemic effects were noted after vaccination with MVA. As expected, pustular skin lesions did develop after Dryvax (Earl et al., 2004).
  Significant adverse events are associated with vaccination with the currently licensed smallpox vaccine. Candidate new-generation smallpox vaccines, such as MVA, produce very few adverse events in experimental animals and in limited human clinical trials conducted near the end of the smallpox eradication campaign. MVA was administered to more than 120,000 individuals in the latter stages of the smallpox eradication campaign without significant adverse events, although the thoroughness of safety data monitoring at that time is unclear. In addition to a vaccination strategy that employs multiple immunizations of MVA, alternative smallpox vaccination strategies may include an initial vaccination with non-replicating virus vaccine followed by a second immunization with a traditional replicating virus vaccine in order to reduce the possibility of vaccine-associate adverse events due to replicating vaccinia virus. Such a scheme of vaccination may be considered as a means of reducing the rate of adverse events associated with traditional smallpox vaccination, provided that vaccine efficacy is not compromised (Meseda et al., 2005).
• Efficacy: Mice immunized intradermally (i.d.) with either 10^8 pfu of MVA, or a prime-boost combination of 10^8 pfu of MVA followed by either 10^6 pfu of Dryvax or 10^8 pfu of MVA survived an intranasal (i.n.) challenge with 25 LD50s of vaccinia virus WR. Furthermore, vaccination with a single dose of 10^8 pfu of MVA resulted in a minimal weight loss (<10%), as did a vaccination combination of 10^8 pfu of MVA followed by 10^6 pfu of Dryvax. When mice that were immunized with a lower dose of 10^6 pfu of MVA were challenged with i.n. vaccinia virus WR, 4/5 survived a challenge with 10 LD50s at either 6 weeks or 12 weeks post-vaccination. When mice received a single immunization of 10^6 pfu of MVA and were challenged with 25 LD50s, 4/5 survived challenge at 6 weeks post-vaccination and 3/5 survived challenge at 12 weeks post-vaccination. In contrast, all animals receiving 10^6 MVA and boosted 6 weeks later with either 10^6 pfu of MVA or 10^6 pfu of Dryvax survived. These results indicate that combinations of MVA are as effective as Dryvax in eliciting immune responses and inducing protective immunity in a mouse model (Meseda et al., 2005).
• Description: The aim of the present study was to compare the immunogenicity and protective ability of MVA (a leading candidate new-generation smallpox vaccine) to the licensed smallpox vaccine Dryvax in a mouse model of vaccination. MVA is a replication-defective vaccinia virus derived from the Ankara strain by more than 500 passages through primary chicken embryo fibroblasts (CEF). This virus grows to high titer in CEF cells but replicates poorly, if at all, in human cells (Meseda et al., 2005).

Mouse Response

• Host Strain: BALB/c, B cell-deficient, and CD1 KO–/– mice
• Vaccination Protocol: Female BALB/c mice (6–10 weeks old, purchased from Frederick Cancer Research Facility, Frederick, MD), B cell-deficient (Taconic Farms), and CD1 KO-/- (CD1KO, from M. Grusby) mice were innoculated with NYVAC at doses from 10^3 to 10^7 pfu. For comparison, and as a positive control, immunization with Wyeth human vaccine strain of vaccinia virus was given by tail scratch (corresponding to skin scratch used for human vaccination). One month after immunization, mice were challenged with 10^6 pfu of WR by intranasal (i.n.) inoculation (Belyakov et al., 2003).
• Persistence: (Belyakov et al., 2003)
• Side Effects: None were mentioned. Replication-defective strains might be valuable as a preliminary immunization to reduce the risk of serious adverse ffects of conventional smallpox vaccination (Belyakov et al., 2003).
• Efficacy: Protection at most doses of NYVAC given i.m. was roughly comparable to that produced by the corresponding doses of MVA given i.m., and no statistically significant difference was detected. It was found that i.m. injection with MVA induced protection of immunized animals in a dose-dependent manner. A dose of 10^7 pfu of MVA given i.m. induced complete protection against challenge with WR (Belyakov et al., 2003).
• Description: At sufficient doses, the protection provided by modified NYVAC replication-deficient vaccinia viruses, safe in immunocompromised animals, was equivalent to that of the licensed Wyeth vaccine strain against a pathogenic vaccinia virus i.n. challenge of mice. A similar variety and pattern of immune responses were involved in protection induced by modified vaccinia Ankara and Wyeth viruses. For both, antibody was essential to protect against disease, whereas neither effector CD4+ nor CD8+ T cells were necessary or sufficient. However, in the absence of antibody, T cells were necessary and sufficient for survival and recovery. Also, T cells played a greater role in control of sublethal infection in unimmunized animals. These properties, shared with the existing smallpox vaccine, provide a basis for further evaluation of these replication-deficient vaccinia viruses as safer vaccines against smallpox or against complications from vaccinia virus (Belyakov et al., 2003).

Monkey Response

• Host Strain: Cynomolgous macaque (Macaca fascicularis)
• Vaccination Protocol: Monkeys were inoculated with 10^8 plaque-forming units (PFU). 24 monkeys were divided into 4 groups: group 1 received an inoculation with 10^8 PFU of MVA at t = 0 and a second 2 months later; group 2 received one injection with 10^8 PFU of MVA followed 2 months later by a standard percutaneous inoculation with Dryvax; group 3 received nothing at t = 0 and 1 Dryvax inoculation 2 months later; group 4 served as the unimmunized control (Earl et al., 2004).
• Persistence: The response to the first MVA inoculation was detected at 1 week, peaked at 2-4 weeks, and was boosted 1 week after the second MVA dose (Earl et al., 2004).
• Side Effects: MVA caused no adverse effects, even when high doses were injected into immune-deficient NHPs (Earl et al., 2004).
• Efficacy: All immunized animals remained clinically well (Earl et al., 2004).
• Description: As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates (NHPs). Here, a highly attenuated MVA is compared with the licensed Dryvax vaccine in an NHP model (Earl et al., 2004).

Monkey Response

• Host Strain: cynomolgus macaques (Macaca fascicularis)
• Vaccination Protocol: Four groups of six captive-bred sub-adult healthy monkeys each were vaccinated: the first group was vaccinated twice with a high dose of 10^8 TCID50 MVA-BN at an interval of 4 weeks, the second group was vaccinated with a low dose of 2 x 10^6 TCID50 MVA-BN followed 10 days later by a s.c. vaccination with Elstree-RIVM, the third group was vaccinated with one s.c. standard dose of Elstree-RIVM, and the fourth group was vaccinated s.c. with one standard dose of Elstree-BN. Group V was sham vaccinated. 15 weeks after the last vaccination, all of the animals were challenged intratracheal (i.t.) with either 10^6 PFU (3 animals per group) or 10^7 PFU (3 animals per group) of MPXV, which were chosen as sub-lethal and lethal challenges, respectively (Stittelaar et al., 2005).
• Persistence: (Stittelaar et al., 2005)
• Side Effects: Elevated body temperatures were observed (Stittelaar et al., 2005).
• Efficacy: All vaccinated animals that were challenged showed an episode of elevated body temperature (>1°C; ~2.65%) that occurred between days 5 and 8 post-challenge which returned to normal by d 12. Only one vaccinated animal developed pocks upon MPXV challenge, while all others showed no clinical signs of the disease apart from an elevated body temperature. This animal, which was vaccinated with MVA-BN (group I), initially developed pocks (>70) on d 11 after the challenge with MPXV (Stittelaar et al., 2005).
• Description: The present study investigated different combinations of candidate and traditional vaccines, followed by MPXV challenge i.t. The MVA strain (MVA-BN, or IMVAMUNE) is currently being tested in >300 human subjects in on-going phase I and II clinical studies, including individuals for whom vaccination with traditional smallpox vaccines is traditionally contraindicated. For the present study, the immune response and efficacy of MVA-BN vaccination were compared to those of a primary vaccination with MVA-BN followed by vaccination with a first-generation smallpox vaccine produced on calf skins (Elstree-RIVM). For this purpose, a low dose of MVA was chosen to prime the immune system, thus reducing the side effects of vaccination with a traditional vaccine shortly thereafter without changing the take rate of the traditional vaccine. In addition, vaccination protocols with Elstree-RIVM alone and vaccination with a second-generation vaccine (Elstree-BN) were evaluated. Elstree-BN is based on the same vaccinia virus strain as Elstree-RIVM, but the former was passaged and produced on chicken embryo fibroblasts to further attenuate the virus and to make a better defined vaccine preparation that does not depend on the use of calves (Stittelaar et al., 2005).

Monkey Response

• Host Strain: Indian rhesus macaques
• Vaccination Protocol: Twenty-five monkeys were enrolled: 6 of the macaques were immunocompetent (groups 1 and 2), and macaques in group 1 were vaccinia naive. Macaques in group 2 had been exposed previously to the attenuated nef- SIVmac239 strain. They were immunized with a single inoculation of NYVAC 1 month before Dryvax vaccination. Group 3 included 7 macaques, 3 that had been infected with the chimeric SHIV 89.6 PD strain for 8 months, 3 that had been infected with the SIVmac251 strain for 12 months, and 1 that had been infected with the nef- SIVmac239 strain for 32 months. Four macaques (group 5) that, at first, had been infected with the same SIVmac251 strain and that subsequently were vaccinated with 3 inoculations of NYVAC, at weeks 10, 19, and 23 after infection (for macaques 480, 644, and H684) or at weeks 42, 48, and 54 after infection (for macaque 3143), were used. The overall time of SIVmac251 infection was 41 months for macaques 480 and 644 and 25 months for macaques H684 and 3143. Four macaques had been infected with SHIV 89.6 PD for 12 months. They were vaccinated with 3 inoculations of NYVAC (10^8 pfu) 6 weeks apart and were challenged with Dryvax 6 months after the final NYVAC immunization. All 25 macaques were vaccinated with Dryvax at the same dose at the times indicated. In brief, the bifurcated needle was immersed in the vaccine suspension and was used to poke the skin 15 consecutive times, in accordance with US Food and Drug Administration (FDA) guidelines. The lesions that developed after smallpox vaccination were photographed every 2 days and were imaged by manually defining the topographic contours of the affected skin (Edghill-Smith et al., 2003).
• Persistence: The immunocompromised macaques were vaccinated with NYVAC at 6 months to a maximum of 36 months before Dryvax challenge, suggesting that this vaccine is able to induce lasting immune responses even as CD4+ helper T cells are progressively depleted. However, the lag between NYVAC and Dryvax vaccinations appears to be important (Edghill-Smith et al., 2003).
• Side Effects: The prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination (Edghill-Smith et al., 2003).
• Efficacy: The prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination, and highlights the importance of neutralizing antibodies in protection against virulent poxvirus (Edghill-Smith et al., 2003).
• Description: The replication competence of live vaccines, such as the only currently available smallpox vaccine, Dryvax, may pose safety concerns when injected in individuals with congenital, acquired, or iatrogenic immunodeficiency. Because the number of patients with immunodeficiency has increased worldwide as a result of the HIV-1 epidemic, the increase in the number of organ transplants, and aggressive chemotherapy in patients with cancer, the risks associated with Dryvax vaccination may affect a larger portion of the population than before. It has been hypothesized that immunization of immunocompromised individuals, with highly attenuated poxviruses, may ameliorate the clinical outcome of Dryvax vaccination. In macaques with modest to severe depletion of CD4+ T cells, it was tested whether immunization with NYVAC before or after infection with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV) would increase the safety of Dryvax vaccination. NYVAC was shown to be safer in severely immunocompromised macaques and that NYVAC priming resulted in a faster resolution of Dryvax-induced lesions in both healthy and immunocompromised macaques (Edghill-Smith et al., 2003).