Immunogenicity and protection of mice following a single vaccination

Although many vaccines are given as a prime and boost, protection after the first dose is desirable particularly during an outbreak. To investigate protection after a single dose of the quadrivalent mRNA-1769 (referred to as mRNA hereafter), we immunized mice IM once with either mRNA or MVA for comparison and bled them 3 weeks later to determine antibody responses (Fig. 1A). Neutralizing antibodies to the M1 and A29 MV proteins are induced by the mRNAs8, whereas antibodies to many proteins induced by MVA may contribute to neutralization in the MV-based assay20. Nevertheless, the neutralizing antibody titer to MVs in the serum at three weeks after the mRNA immunization was a log higher than that achieved with MVA (Fig. 1B). The Western Reserve (WR) strain of VACV has been extensively used for studies of pathogenicity and a lethal dose 50 (LD50) of 105.3 was determined for IN inoculation of DBA strain mice21. Here we used a VACV WR recombinant (WRvFire) that expresses firefly luciferase to enable non-invasive imaging of infected BALB/c mice22. The severity of infection of control BALB/c mice was related to virus dose leading to the loss of 30% or more of starting weight (required trigger for euthanasia) or death in 0 of 6 mice at 104 PFU, 2 of 6 at 105 PFU, and 6 of 6 at 106 PFU (Fig. 1C), resulting in a calculated LD50 of 105.1. In contrast, mice receiving a single injection of mRNA or MVA vaccine exhibited only minor weight loss and 100% survival even at the highest challenge dose (Fig. 1C).

Fig. 1: Single immunization protects against intranasal infection.

A BALB/c mice were divided into 9 groups (n = 6 per group) and mock immunized with PBS or immunized with 107 PFU of MVA or 8 µg of mRNA. The mice were bled on day 0 and 21 prior to IN challenge with 104, 105, or 106 PFU of VACV WRvFire. B Anti-VACV MV neutralization titers expressed as NT50 of all animals receiving PBS, mRNA or MVA. Dots represent indivual animals, bars represent geometric mean titers. LOD, limit of detection. C Mice were weighed daily and % of starting weight and survival plotted for each group. Error bars represent standard error of the mean (SEM). D Luciferin was injected IP on days 2, 4, 7 and 11. Bioluminescence (BL) is depicted by a pseudocolor scale with red representing highest and blue lowest intensity. Each row represents an individual mouse imaged on successive days. E Photon flux was determined for head and body (chest and abdomen) region of interest (ROI). Geometric mean photon flux is indicated by bars. Significant differences between mRNA and MVA neutralization titers and photon flux for days 2 and 4 were evaluated by Mann–Whitney test. **p < 0.01, **** p < 0.0001.

Bioluminescence (BL) imaging was used to determine virus replication at the site of IN inoculation and spread in control and immunized mice following IP administration of the firefly luciferase substrate luciferin on successive days following challenge (Fig. 1D). Because firefly luciferase decays with a half-life of ~2 h in living cells23, BL reflects enzyme activity close to the time of analysis. Luker and Luker24 demonstrated a strong correlation between BL and plaque-forming units using a recombinant VACV similar to ours. In control mice challenged with 104 PFU of which all survived, BL in the heads of mice reached a peak on day 7 and diminished by day 11; after challenge with 105 PFU, BL was also detected in the chest by day 4 and was diminished in surviving mice by day 11; after challenge with 106 PFU for which there were no survivors, BL was detected in the chest on day 2 and extended to the abdomen by day 4 (Fig. 1D). We concluded that weight loss is associated with upper respiratory infection and that severe disease coincides with spread to the lungs and abdominal organs in control mice. BL was reduced in the heads of immunized mice and few exhibited BL in the chest and none showed BL in the abdomen (Fig. 1D). However, the BL in the heads of mice immunized with mRNA and challenged with 106 PFU of VACV appeared more intense on day 4 than in mice immunized with MVA.

To avoid image saturation, a relatively short exposure was used for Fig. 1D, and therefore could not reveal low levels of virus replication. Photon flux, however, provides a wide dynamic range and quantitative measurement of BL in regions of interest (ROI). Analysis of the head region confirmed high levels of virus replication in control mice at all challenge doses (Fig. 1E). The immunized mice exhibited a progressive increase in photon flux in the head with increasing challenge dose, but significantly lower than in control mice with return to baseline in all animals by day 7. Photon flux values above baseline in the bodies (which included the chest and abdomen) of immunized mice were detected only with the 106 PFU challenge and were transient (Fig. 1E). However, the photon flux was significantly greater in the heads of mRNA single-immunized mice than in the MVA single-immunized mice on days 2 and 4 and in the bodies on day 4. While neither MVA nor mRNA provided sterilizing protection at the IN site of inoculation after a single shot when challenged with the potentially lethal 106 PFU dose of VACV, replication was greatly reduced and transient in the head and bodies.

Relationship of the dose of a single mRNA immunization with neutralizing antibody and protection

Groups of mice were immunized once with 0.5 µg, 2.0 µg, or 8.0 µg of mRNA. Serum was obtained at 3 weeks in each case, and also at 1 week for the 8.0 µg dose (Fig. 2A). The 3-week 50% neutralization titers (NT50) values were proportional to dose with statistical significance between the low and high doses. The titer for the 8.0 µg dose at one week was similar to the 2.0 µg dose at 3 weeks (Fig. 2B).

Fig. 2: Effects of single mRNA dose and interval before challenge on protection.

A Groups of BALB/c mice (n = 5) were vaccinated and challenged after 1 or 3 weeks with VACV via intranasal (IN) administration. B Anti-VACV MV neutralization titers expressed as NT50 determined 1 week after mock immunization with PBS or 8 µg of mRNA and 3 weeks after immunization with 0.5, 2.0, and 8 µg of mRNA. Individual animals represented by dots and geometric mean of the group represented by each bar. C Mice inoculated IN with 106 PFU of VACV WRvFire at 3 weeks after vaccination with 0.5, 2.0, or 8.0 µg of mRNA were weighed daily and percent of starting weight plotted. Error bars represent SEM. D Mice inoculated IN with 106 PFU of WRvFire at 1 week after vaccination with 8 µg of mRNA were weighed daily and percent of starting weight plotted. Error bars represent SEM. E Mice challenged at 3 weeks following vaccination were imaged following injection of luciferin on days 2, 4, 8, and 11. Bioluminescence is depicted by a pseudocolor scale with red highest and blue lowest intensity. Each row represents an individual mouse imaged on successive days. Photon flux was determined for head and body ROI for individual animals, with bar representing the geometric mean of each group. F Same as preceding panel except that mice vaccinated with 8 µg of mRNA and challenged after 1 week were imaged on days 2, 4, and 7. Significance for panels B and E were determined by Kruskal–Wallis test with Dunn’s post-hoc multiple comparisons and for panel F by Mann–Whitney test. *p < 0.05, **p < 0.01.

When challenged with 106 PFU of WRvFire, the control mice rapidly lost weight and succumbed to the infection, whereas the immunized mice all survived. The mice immunized with 0.5 µg of mRNA lost about 20% of their weight when challenged at 3 weeks, while the mice receiving 2 µg of mRNA lost about 6% (Fig. 2C). Mice that received 8 µg of mRNA retained their starting weight when challenged at 3 weeks (Fig. 2C) and lost little weight when challenged at 1 week (Fig. 2D).

The mice were imaged over a period of 11 days. As in the previous experiment, the phosphate buffer saline (PBS) control mice showed high BL in the head and extensive spread of the infection to the chest and abdomen (Fig. 2E). Mice immunized with 0.5 µg mRNA exhibited strong BL in the head and some in the chest, which largely cleared by day 8 (Fig. 2E) consistent with the change in weight (Fig. 2C). Mice immunized with 2 or 8 µg of mRNA exhibited low BL in the head, and none detected in the chest or abdomen (Fig. 2E).

Photon flux measurements demonstrated significantly lower BL in the heads and bodies on days 2 and 4 of mice immunized with 2 or 8 µg of mRNA compared to the control, but the difference between 2 and 8 µg was not statistically significant (Fig. 2E). Diminution of the photon flux of some mice immunized with only 0.5 µg of mRNA was delayed and the difference from the control did not reach significance on days 2 and 4. The BL and photon flux of mice immunized with 8 µg mRNA and challenged after one week was most similar to that of mice immunized with 2 µg mRNA and challenged at 3 weeks (Fig. 2F).

In summary, three weeks after a single vaccination, NT50 values of 102 to 103 achieved with 2 µg or higher mRNA resulted in little or no weight loss and reduced virus replication upon IN challenge with 106 PFU of VACV. A similar result was achieved 1 week after a single 8 µg mRNA vaccination. Taken together these data indicated rapid protective immunity following a single mRNA vaccination that corresponded with vaccine dose and neutralizing antibody titers.

Increased immune response and sustained protection following a boost vaccination

To determine the impact of boosting, mice received a second inoculation of 8 µg of mRNA or 107 PFU of MVA at 3 weeks after the first (Fig. 3A). For each vaccine, neutralizing antibody increased significantly after the boost and decreased slightly between 3 and 16 weeks (Fig. 3B). Again, the NT50 titers achieved with mRNA were about a log higher than with MVA.

Fig. 3: Enhanced protection after second immunization.

A BALB/c mice were divided into 6 groups (n = 6) and mock immunized with PBS or immunized with 107 PFU of MVA or 8 µg of mRNA on days 0 and 21 and challenged IN on weeks 3 and 16 with VACV WRvFire. B Anti-VACV MV neutralization titers for individual animals are expressed as NT50 with bars representing the geometric mean of each group. C Comet spread assay. Pooled serum diluted 1:50 was added to BS-C-1 cell monolayers at 1 h after infection with VACV strain IHD-J and stained with crystal violet after 48 h incubation at 37 °C. D Percent of starting weights and survival are shown as the mean of each group for each day with error bars representing SEM. E BL on days 2, 4, 7, and 10. Each row represents an individual mouse imaged on successive days. F Photon flux of head and body ROI for individual animals, with bar representing the mean of each group. Significance was evaluated by Kruskal–Wallis test with Dunn’s post-hoc multiple comparisons tests in panel (B) and by Mann–Whitney test in panel (F). **p < 0.01. ***p < 0.001, ****p < 0.0001.

As stated earlier, the NT50 titers refer only to neutralization of MVs. A qualitative assay to measure functional antibodies targeting the EV was conducted as follows. Antibodies to the VACV homologs of the EV proteins A35 and B6 reduce virus spread on a cell monolayer when antibody is added after virus adsorption and the plates are overlaid with liquid medium18. After incubation for 2 days, crystal violet staining revealed satellite plaques with a characteristic comet-like distribution in the presence of non-immune serum, which were reduced partially by 3-week serum and more completely by 6-week serum (Fig. 3C). The extent of comet inhibition decreased slightly with the serum obtained at 16 weeks (Fig. 3C).

The PBS-inoculated mice lost weight and 5 of the 6 succumbed when challenged IN with 106 PFU of WRvFire, whereas mRNA- and MVA-immunized mice lost little or no weight and survived when challenged at 6 or 16 weeks (Fig. 3D). Although the MVA vaccine induced lower neutralizing antibodies than mRNA, it may have exceeded the threshold necessary for protection of mice.

Images obtained after the 6-week challenge revealed transient BL in the heads of 2 of the 6 mice that received MVA and none that received mRNA (Fig. 3E). BL was not detected in the bodies of immunized mice at week 6 (Fig. 3E). The mean photon flux levels in the heads of immunized mice were 3 and 4 logs lower than controls in MVA- and mRNA- immunized mice, respectively (Fig. 3F). Additionally, the photon flux values diminished more rapidly in the mRNA boosted mice than in mice that received only a single immunization (compare Fig. 3F and Fig. 1E). The mean photon flux in the bodies of mRNA-immunized mice were at baseline. On day 2, the photon flux in the bodies of MVA-immunized mice was significantly higher than in mRNA-immunized mice but within 2 days dropped to baseline. When challenged at 16 weeks, BL was transiently detected in the chest of one MVA-immunized mouse and a few in each group had transient BL in the head (Fig. 3E). The mean photon flux of the heads was higher than at 6 weeks but still nearly 3 logs lower than controls (Fig. 3F). The photon flux in the bodies was only slightly above baseline on day 2 before diminishing to baseline (Fig. 3F). Thus, a high degree of protection was sustained for at least 4 months.

Protection against MPXV

Wild-derived inbred castaneous/EiJ (CAST) mice are more susceptible to MPXV and other orthopoxvirus compared to BALB/c and other common inbred mouse strains25. Nevertheless, CAST mice make strong IgG and T cell responses; their susceptibility to orthopoxviruses appears to be largely due to low numbers of natural killer cells26,27. In the next experiments, CAST mice were primed and boosted with 1 or 4 µg of mRNAs (Fig. 4A). The anti-VACV neutralizing titers of the mice primed with 4 µg of mRNA were significantly higher than the titers of mice immunized with 1 µg of mRNA or 107 PFU of MVA (Fig. 4B). After boosting, the anti-VACV titers obtained with 1 and 4 µg of mRNA were both significantly higher than that obtained with MVA, but were not significantly different from each other. The anti-MPXV titers were about a log lower than the anti-VACV titers but here also the titers of mice boosted with 1 or 4 µg of mRNA were significantly higher than those that received MVA (Fig. 4C).

Fig. 4: Protection of CAST mice challenged intranasally and intraperitoneally with MPXV.

A Groups of CAST mice (n = 12) were primed and boosted with PBS, 107 PFU of MVA, or 1 or 4 µg of mRNA before challenge IN or IP with MPXV. B Anti-VACV MV neutralization titers expressed as NT50 for individual animals with bars representing the geometric mean of each group. C Anti-MPXV MV neutralization titers expressed as NT50 for individual animals with bars representing the geometric mean of each group. D Six mice from each group were challenged IN with 105 PFU of MPXV and survival determined as percentage of surviving mice on each day of study. E Six mice from each group were challenged IP with 104 PFU of MPXV and survival determined as percentage of surviving mice on each day of study. Significance for panels B and C were analyzed by Kruskal–Wallis with Dunn’s post-hoc multiple comparisons tests and for panels (D) and (E) by log-rank (Mantel–Cox) survival test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Previously, we determined that 5- to 7-weeks old CAST mice all succumbed to IN and IP challenges with 105 and 104 PFU of MPXV USA-2003, respectively28. In the present study, we used the same challenge doses, though the mice were 6 weeks older due to the prime and boost immunization protocol and more resistant to lethal infection. Three of the six control PBS mock-immunized mice succumbed to the 105 PFU IN infection within 8 days, whereas all of the immunized mice survived for at least 2 weeks, a difference that was statistically significant (Fig. 4D). Four of the six mice challenged IP with 104 PFU of MPXV succumbed while all immunized mice survived, which was also statistically significant (Fig. 4E). The similar protection with the 1 and 4 µg doses of mRNA was consistent with their similar induced neutralization titers. MVA also protected animals from lethality, even though the neutralization titers were relatively low.

Protection against intrarectal (IR) and percutaneous infections

Although MPXV can spread through the respiratory route, human-to-human transmission occurred mostly through male-to-male sexual activity during the recent global outbreak29, and lesions in the rectal mucosa were common and confined to that area in the majority of cases30. Additionally, there is recent evidence of sexual spread of the more pathogenic clade I MPXV in Africa31. It was therefore pertinent to determine whether mRNA-administered IM would protect against IR and percutaneous challenges. Mice were primed and boosted with mRNA or MVA vaccines and then inoculated with 106 PFU of WRvFire IR. In control mice, local BL was detected on day 2, increased on day 4 with detectable BL in the abdominal region of one mouse, and was diminished by day 8 (Fig. 5A). In contrast, no virus replication was detected by either BL (Fig. 5A) or more sensitive photon flux measurements (Fig. 5B) in immunized mice.

Fig. 5: Protection from intrarectal and percutaneous infections.

A BL of mice (n = 5 per group) following intrarectal infection with 106 PFU of VACV WRvFire. Each row represents an individual mouse imaged on successive days. B Total photon flux of the rectal area for individual animals with bars representing the geometric mean of each group. C BL of mice (n = 5 per group) following percutaneous infection with 105 PFU of VACV WRvFire. D Total photon flux of the area around the tail for individual animals with bars representing the geometric mean of each group.

The ACAM2000 smallpox vaccine was administered percutaneously in humans and caused a pustular lesion within 7 to 8 days. Here we used a similar inoculation strategy with mice except that the pathogenic VACV WRvFire was used instead of the viral vaccine strain. Following percutaneous inoculation of control mice with 105 PFU of WRvFire, BL peaked on days 4 to 7 at the site of inoculation and substantially declined by day 11 (Fig. 5C, D). In contrast, no BLI or increased photon flux was detected in immunized mice. Thus, IM inoculation with either mRNA or MVA protects against IN, IR, and percutaneous infections in VACV mouse models.

Passive immunization protects against subsequent virus infection

Previous studies indicated that antibody has a dominant role in protection mediated by live VACV vaccines32,33,34. It was of interest, therefore, to determine whether mice would be protected from VACV challenge by passive transfer of serum from mRNA-immunized animals. Because only small amounts of mouse serum were available, we used serum from cynomolgus macaques that was collected two weeks after priming and boosting with mRNA, ACAM200035, or ACAM300036 (Fig. 6A). ACAM2000 and ACAM3000 are vaccines that consist of replication-competent and replication-defective (MVA), respectively. In a preliminary experiment we determined that the titer of macaque neutralizing antibody in the blood of mice (n = 4) had a half-life of 7 days following IP inoculation. For the challenge experiment, mice were injected IP with pooled serum from macaques that were mock immunized with PBS or vaccinated with 15, 50, or 150 µg of mRNA or the recommended human doses of ACAM2000 or ACAM3000. The NT50 titers of the sera from mice at 1 day after injection of the macaque sera were low after transfer of ACAM2000 and ACAM3000 immune serum and only slightly higher with 15 µg mRNA but were significantly higher after transfer of the 50 and 150 µg mRNA serum (Fig. 6B). The titers of the sera at 12 days were similar to each other (Fig. 6B) due to antibody induced by the challenge virus as will be discussed below.

Fig. 6: Immune serum protects against subsequent virus infection.

A Immunization and challenge scheme. Macaques were immunized by priming and boosting with PBS, ACAM2000, ACAM3000, or 15, 50, or 150 µg of mRNA vaccine. Pooled serum was injected IP into BALB/c mice (n = 5 per group) and one day later the mice were challenged with 105 PFU of VACV WRvFire. B Serum was obtained from mice 1 day post macaque serum transfer and 12 days post VACV challenge and the anti-VACV MV neutralization titer was determined for each animal with the bar representing the geometric mean of each group. C Mice were weighed daily following challenge and the mean of the group is reported each day with error bars representing SEM. Color key is same as in following panel. D Survival curves are shown for each group of mice with the percent surviving mice reported each day post challenge. E Relationship of weight loss to NT50 is plotted for each individual animal. R2 of 0.79 was determined by linear regression. F BL obtained on days indicated. Each row represents an individual mouse imaged on successive days. G Total photon flux was determined for head and body ROI for individual animals, with bar representing the geometric mean of each group. Signifcance was evaluated by Kruskal–Wallis test with Dunn’s post-hoc multiple comparisons tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Panel A was created in https://BioRender.com.

The mice were challenged IN with 105 PFU of VACV WRvFire (1 log less than after active mRNA immunizations) at one day after IP injection of serum. Mice injected with serum from control macaques that received PBS or had been immunized with ACAM3000 rapidly lost weight and all succumbed (Fig. 6C, D). Mice that received the ACAM2000 or the 15 µg mRNA macaque serum also exhibited severe weight loss, but 3–4 of the 5 mice recovered before losing 30% of their weight. In contrast, all mice that received sera from the macaques immunized with 50 or 150 µg mRNA survived, although the former suffered more weight loss before recovering. There was an inverse relationship between the neutralization titers on day 1 and maximum weight loss of surviving mice (Fig. 6E). Nevertheless, the mice receiving macaque immune serum were not as well protected against weight loss as mice that were actively immunized even though the NT50 values were in the same range at the time of challenge. Several possible explanations for the difference are considered in the Discussion.

Mice injected with the macaque control (PBS) serum exhibited intense BL in the head and chest as did mice that received the ACAM2000 or ACAM3000 serum (Fig. 6F). Mice that received the 15 µg mRNA serum also showed strong BL in the head and detectable BL in the chest, although lower than in the mice that received non-immune serum (Fig. 6F). However, the mice that received the 50 and 150 µg mRNA sera exhibited much lower BL in the head and undetectable BL in the body. The photon flux measurements in the heads and bodies of mice that received the 50 µg and 150 µg mRNA macaque sera were significantly lower on day 7 than that of mice that received the control and ACAM3000 (MVA)-immune sera (Fig. 6G).

Although the mice receiving control serum succumbed quickly following challenge, it was possible that an immune response to the challenge virus contributed to the protection of mice receiving immune serum. To investigate this possibility, we determined the neutralizing antibody titers of the surviving mice on day 12 (Fig. 6B). The increase in NT50 was 4- to 12-fold for surviving mice that received the ACAM 2000 serum, 6- to 13-fold for the 15 µg mRNA serum and less than twofold for the 50 µg mRNA serum. The NT50 decreased for the 150 µg mRNA serum consistent with suppression of the infection and the 7-day half-life of the macaque antibodies. Thus, an immune response to the challenge virus was unlikely to benefit mice that received the 150 µg serum, though it may have helped those that received lower titer macaque serum.

Passive immunization provides protection post-exposure to VACV infection

The VACV infection model uses the purified MV form of the virus for challenge. However, spread of VACV within an animal depends on the EV form of VACV with different surface proteins37. To determine whether the immune serum could control spread of an established infection, the same macaque serum used in Fig. 6 was added one day after the virus inoculation (Fig. 7A). Mice that received the 150 µg mRNA serum exhibited transient weight loss followed by recovery on day 7, whereas 4 of 5 mice that received nonimmune serum succumbed to the infection (Fig. 7B, C). The recovery of the immunized mice was also demonstrated by the diminution of BL in the head between day 4 and 7 (Fig. 7D). Photon flux measurements at the day 7 peak in the heads and bodies of mice that received immune serum were significantly lower compared to mice that received control serum (Fig. 7E). Taken together, passive antibody elicited by mRNA-1769 vaccination provided protection when administered before or after challenge.

Fig. 7: Immune serum protects against prior virus infection.

A Immunization and challenge scheme. Macaques were immunized by priming and boosting with PBS or 150 µg of mRNA with one month between doses and peak immune serum taken two weeks post boost. Mice (n = 5 per group) were infected IN with 105 PFU of VACV WRvFire and pooled macaque serum was inoculated IP one day later. B Individual mice were weighed daily following challenge with error bars representing SEM. C Survival curves are shown for each group of mice with the percent surviving mice reported each day post challenge. D BL obtained on days indicated. Each row represents an individual mouse imaged on successive days. E Total photon flux was determined for head and body ROI for individual animals, with bar representing the mean of each group. Significance was evaluated by Mann–Whitney test. **p < 0.01. Panel A was created in https://BioRender.com.

Passive immunization protects against acute disease in the absence of an adaptive immune response

The recovery of weight at day 7 following virus challenge (Fig. 6C) raised the possibility of a role for an immune response elicited by the infection, although protection was shown to correlate with the abundance of neutralizing antibody transferred. To investigate a role for adaptive immunity, we passively transferred control nonimmune or 150 µg mRNA immune serum to C57Bl/6 Rag2 (recombination activating gene 2) knock-out (KO) mice, which have a deletion of the entire RAG2 protein coding region and consequently produce no mature T cells or B cells38. As controls, immunocompetent parental C57Bl/6 mice also received control and immune serum. The passively administered neutralizing antibody titers were similar in both mouse strains as measured 1 day post transfer (Fig. 8A). Following challenge with 105 PFU of WRvFire, the C57Bl/6 and Rag2 KO mice that received the control serum rapidly lost weight and succumbed to the infection (Fig. 8B, C). By comparison, Rag2 KO and C57Bl/6 mice that received the immune serum lost relatively little weight and all survived until the experiment was ended at 28 days. A previous study found that BALB/c SCID mice passively immunized with rabbit polyclonal anti-L1, -A33 and -B5 antibodies had a 50% mean survival time of 26 days following IN challenge with VACV18.

Fig. 8: Immune serum protects immunodeficient Rag2 KO mice.

A Anti-VACV MV neutralization titers of serum from mice (n = 3) at 1 day after injection of macaque serum. Bars represent the geometric mean of each group. B Mice were challenged one day after receiving control or immune serum and weighed daily and the mean weight of the group is reported eact day with error bars representing SEM. C Survival curves are shown for each group of mice with the percent surviving mice reported each day post challenge. D BL shown for days indicated. Each row represents an individual mouse imaged on successive days. Non, non-immune; Im, Immune. E Total photon flux was determined for head and body ROI for individual animals, with bar representing the mean for each group. Significance was evaluated by Kruskal–Wallis with Dunn’s post-hoc multiple comparisons tests. *p < 0.05, ns, not significant.

Live imaging provided information regarding virus replication. BL was intense in the heads of mice that received control serum and was also detected in their chests, whereas the BL was less intense in the heads and undetectable in the bodies of Rag2 KO and C57Bl/6 mice that received immune serum (Fig. 8D). Furthermore, the mean photon flux was up to 2 logs lower in the heads of mice that received immune serum compared to nonimmune serum but there was no significant difference on combined 7 + 8 days between the Rag2 KO and C57Bl/6 mice that received the immune serum. The photon flux in the bodies of the passively immunized mice was barely above the baseline and there was no significant difference between the Rag2 KO and C57Bl/6 mice (Fig. 8E). However, virus was cleared from the head of the immunocompetent mice by day 11, whereas in the Rag2 KO mice luminescence was still detected on day 28, indicating the presence of live virus (Fig. 8E).

The conclusions of the passive transfer experiments are that antibody is sufficient to protect immunocompetent and immunodeficient mice from acute disease but that an adaptive immune response contributes to virus clearance.