Feline immunodeficiency virus (FIV) vaccine efficacy and FIV neutralizing antibodies

2.1. Animals

Specific pathogen free (SPF) cats were purchased from Liberty Research, Inc. (Waverly, NY), Harlan Sprague Dawly, Inc. (Madison, WI), Cedar River Laboratories (Mason City, IO), or were bred in the Laboratory of Comparative Retrovirology & Immunology at the University of Florida. Based on the Institutional Animal Care and Use Committee (IACUC) policy to minimize the animal use, Study 8 used the vaccinated/protected cats from Study 3 (Group 3B). Cats challenged with FIV_FC1 (subtype-B) were 8 weeks of age, while all other cats were 12–16 weeks old. All cats were maintained and utilized according to the policy and protocols approved by IACUC.

2.2. Immunization

The Fel-O-Vax^® FIV vaccine is composed of 1.5×10⁷–2.5×10⁷ inactivated infected Fet-J cells plus 50 μg of inactivated whole viruses (IWVs), while the prototype FIV vaccine consisted of 500 μg IWVs (FIV_Pet plus FIV_Shi) at a 50/50 ratio of each strain, supplemented with cytokine(s) [5]. Both vaccines were formulated in the FDAH’s (FD-1) adjuvant [5,20]. All prototype IWV vaccines were supplemented with one of the following cytokines or cytokine combinations at 5 μg/dose (Tables 1 and 2): human interleukin-12 (HuIL-12; Genetics Institute, Cambridge, MA), feline IL-12 (IL-12; R&D Systems, Minneapolis, MN), and feline IL-15 (IL-15) produced by our laboratory [21]. Some studies supplemented Fel-O-Vax^® FIV with IL-12 and IL-15. The SPF cats received subcutaneous (SC) immunizations alone, or in combination with intradermal (ID) immunizations (SC/ID), in 3-week intervals.

2.3. FIV inoculum and challenge

Challenge viruses were classified into tiers of 1, 2, or 3 based on a previously defined HIV-1 tiering system [19]. Moreover, tier-1 FIVs are homologous vaccine strain(s) or those from the same subtypes as vaccine strain(s) that are readily neutralized by vaccine-induced NAbs. Tier-2 FIVs are those from subtypes same as vaccine strain(s) but more resistant to vaccine-induced NAbs, while tier-3 FIVs are those from subtypes different from vaccine strain(s) and are highly resistant to vaccine-induced NAbs. Hence, FIVs from tiers 2 and 3 are the most difficult to protect.

All in vivo-derived inoculums were serially transferred from one cat to another, in a total of one-three cats, before final blood samples were collected from cats at peak viremia [17,18]. The in vivo-derived FIV_Bang (recombinant subtype-A/B) and FIV_UK8 (subtype-A) consisted of cryopreserved pooled plasma from infected cats, while the in vivo-derived FIV_FC1 (subtype-B) and FIV_NZ1 (recombinant subtype-F′/C) were cryopreserved pooled PBMC from infected cats. The in vitro-derived FIV_Pet consisted of culture fluid (<16 culture-passages) from the PBMC of infected cats as described [17]. The vaccinated and control cats were challenged intravenously (IV) 3–4 weeks following the final immunization [17,18].

2.4. Monitoring for FIV infection, T-cell status, and NAbs

Challenged cats were considered positive for FIV infection when testing positive by immunoblot analysis, virus isolation, proviral PCR, and by a decrease in CD4⁺ T-cell counts and/or CD4⁺/CD8⁺ T-cell ratios [17,18]. Proviral PCR utilized primers targeting conserved FIV env sequences under previously described conditions [22]. The sequences were 5′-GAAATGTATAATATTGCTGG as forward primer and 5′-TTACATCCTAATTCTTGCATAG as reverse primer. The approximate amount of proviral DNA per cell was determined via semi-quantitative PCR using varying dilutions from a known number of cells as described [23]. These parameters were performed on PBMC at 3–4, 6, 9, 12, 16, and 20–24 weeks post-challenge (wpc), or monthly thereafter until 52 wpc. Virus isolation was also performed on tissues at 20–52 wpc. Cats were deemed protected if testing negative for all the above parameters following vaccination and challenge. Since vaccine-induced FIV antibodies were already present, cats were considered antibody positive for infection when the antibodies to the FIV p24 and Env gp95/100 were enhanced, or remained elevated after challenge [17,18]. NAb analysis was performed as described for HIV-1 [24] with modifications. Briefly, heat-inactivated (56°C, 45 min) cat sera using the in vitro-derived FIV inoculums (<16 passages) were obtained as previously described [17,18,25] and varying serum dilutions were reacted with appropriate challenge virus for 1 hour at 37° C. These mixtures were then added in vitro to naïve feline PBMC taken from SPF cats, and culture supernatant was assessed for virus every 3 days over a 15 day period.

2.5. Passive-transfer studies

One passive-transfer study (Study PT1) was performed with heat-inactivated, pooled unpurified sera from Fel-O-Vax FIV-vaccinated SPF cats or from non-vaccinated/age-matched cats [25]. Each cat received cross-matched compatible serum equivalent to 30% of the recipient’s total blood volume. Due to the large volume, cats received a 20% volume in the first IV transfer on day -1, with FIV challenge on day 0, followed by a 10% volume in the second transfer and monitored for infection for 24 weeks. Another two sets of SPF cats (Studies PT2 and PT3) similarly passively transferred with 6.5 mg immunoglobulin per kg body weight of purified pooled antibodies from either vaccinated or PBS-immunized SPF cats in the first transfer and 3.5 mg/kg in the second transfer. Antibodies purified by using caprilic acid purification followed by ammonium sulfate precipitation [26] had an IgG purity of >95% based on gel analysis (data not shown).

2.6. Statistics

Statistically significant differences were determined by the pair-wise Mann-Whitney Rank Sum test (SigmaPlot version 11.0, San Jose, CA), and Bonferroni’s correction was also performed in Figure 2. All analyses were considered statistically significant when p<0.05.

3.1. Vaccine protection against heterologous-subtype viruses

In the short-duration Studies 1 and 2 against the recombinant subtype A/B FIV_Bang isolate (Table 1), statistically significant protection was observed with the prototype dual-subtype IWV (Groups 1A+2A vs. Groups 1B+2B, p=0.001) but not with the dual-subtype Fel-O-Vax^® FIV. Normal CD4⁺ T-cell counts also confirmed this protection in the IWV-vaccinated/protected cats which were statistically different from the infected control cats (Fig. 2A, p<0.001).

In another set of short-duration studies (Table 1, Studies 3 and 4), significant protection was observed with both Fel-O-Vax^® FIV (Study 3, p<0.001) and prototype IWV (p=0.037) against the heterologous (non-vaccine) subtype-B FIV_FC1. FIV_FC1 belongs to heterologous-subtype B, unlike the recombinant FIV_Bang which is more closely related to the homologous (vaccine) subtype-A viruses except for the subtype-Benv_V3-V9 [10]. Hence, the FIV_FC1 challenge is considered a more rigorous challenge in comparison to the subtype-A/B FIV_Bang. The combined result of Fel-O-Vax^® FIV and IWV demonstrated high protection against FIV_FC1 (Groups 3A+3B+4A+4B vs. Groups 3C+4C, p<0.001). This result was also supported by normal CD4⁺ T-cell counts and CD4⁺/CD8⁺ T-cell ratios (Fig. 2C and 2D) which were statistically different from the values of the infected control group (CD4⁺ T-cell counts, p=0.015; CD4⁺/CD8⁺ ratios, p=0.001).

In the study using a recombinant-subtype virus from New Zealand (FIV_NZ1) (Table 1, Study 5), both the IWV (6 of 8) and the Fel-O-Vax^® FIV (2 of 5) conferred some protection, but the protection rate of only IWV group was statistically different from the infected control group (p=0.008). FIV_NZ1 belongs to subtype-F′/C and has no subtype homology to either vaccine viruses (Fig. 1). The CD4⁺ T-cell counts and CD4⁺/CD8⁺ ratios of the vaccinated/protected groups were similar to those of the unchallenged control group but significantly higher than those of the infected control group (Fig. 2E and 2F, p<0.001).

3.2. Vaccine efficacy against homologous subtype-A strains

The next study determined whether the variation of vaccine efficacy observed against heterologous-subtype viruses also existed amongst the challenge strains from the same (homologous) subtype as the vaccine strain (FIV_UK8; Table 1, Study 6). The IWV afforded a significant combined protection rate of 40% (8 of 20) against challenge with the subtype-A FIV_UK8. Increasing the frequencies of vaccination from 3X (6 of 15) to 6X (2 of 5) did not affect the 40% protection rate. The vaccinated/protected cats had normal CD4⁺ T-cell counts and CD4⁺/CD8⁺ ratios which were significantly higher than those of the infected controls (Fig. 2G and 2H, both p<0.001). In contrast to the FIV_UK8 study, the IWV vaccine conferred 100% protection against the in vitro-derived homologous-strain FIV_Pet (Table 1, Study 7) which showed an efficacy identical to those against in vivo-derived FIV_Pet [17].

3.3. Long-duration efficacy against heterologous-subtype viruses

A long-duration efficacy study with the Fel-O-Vax^® FIV was performed with a slightly higher FIV_FC1 challenge dose than Study 3 to ensure complete infection in all (>1 year old) cats (Table 2). The eight protected cats from Group 3B of Study 3 (after one-year confirmation of FIV-negative status) were given a one-year boost and subsequently challenged with FIV_FC1 (Table 2, Groups 8A). An additional five cats (3X vaccinated one-year earlier without challenge, Group 8B) received a one-year boost, and were challenged 3 weeks later.

Six of 8 vaccinated/previously-protected cats in Group 8A were protected again at a slightly-higher FIV_FC1 challenge dose, while all five previously-vaccinated/challenge-naive Group 8B cats and all five PBS-immunized cats became infected. Four of 6 protected cats belonged to Group 8A1, which were boosted with the Fel-O-Vax^® FIV/IL-12+IL-15, while the remaining two protected cats from Group 8A2 were boosted with Fel-O-Vax^® FIV alone. This suggests that the cytokines in the one-year boost enhanced vaccine protection (Group 8A1 vs. Group 8A2+8B, p=0.046).

In another long-duration study (Table 2, Study 9), 2 of 4 cats vaccinated 3X with the Fel-O-Vax FIV^® in the first year (without challenge), followed by a one-year boost, were protected against the in-vivo-derived FIV_Bang at the same challenge dose and timing as Study 1.

3.4. Vaccine-induced NAbs as the mechanism of vaccine protection

The importance of vaccine-induced NAbs in the prophylactic protection observed above were next evaluated. Cats vaccinated 3X with IWV or Fel-O-Vax^® FIV (before challenge) developed strong NAb titers to FIV_Pet and FIV_Shi, while substantially less to FIV_Bang and minimal-to-no titers to FIV_UK8, FIV_FC1, and FIV_NZ1 (Table 3). After three vaccinations the Fel-O-Vax^® FIV (without cytokines) induced NAbs in more cats than the IWV, except for the NAbs to FIV_Pet and FIV_Shi .

The vaccine protection against the homologous FIV_Pet (Table 1, Study 7) correlated with vaccine-induced NAb titers to FIV_Pet (Table 3), while no such correlation was observed against the subtype-A FIV_UK8 (Tables 1 and 3). Although both vaccines induced minimum-to-no NAb titers to heterologous subtype-F′/C FIV_NZ1 (Table 3), 8 of 13 (62%, combined result) vaccinated cats were protected against FIV_NZ1 (Table 1, Study 5). Furthermore, both vaccines conferred 75–100% protection against a heterologous-subtype FIV_FC1, even though these vaccines induced minimal-to-no anti-FIV_FC1 NAb titers (Tables 1 and 3). In contrast, varying anti-FIV_Bang NAb titers (<10–1000) were induced by vaccination (Table 3, only average titers), although these titers did not correlate with the protection observed against the subtype-A/B FIV_Bang (individual data not shown).

The addition of IL-12 and IL-15 to the Fel-O-Vax^® FIV did not significantly enhance the magnitude or frequency of the NAb titers to heterologous-subtype strains following the initial vaccinations or the annual boost (Table 3). However in long-duration Study 8 (Table 2), the vaccine/cytokines group had a significantly higher protection rate compared to the non-cytokine group, suggesting that these cytokines may enhance vaccine protection memory without enhancing the NAb titer.

3.5. Vaccine-induced NAbs to define FIV tiers

Based on the definition of virus tiers modified from those described for HIV-1 [19], both vaccines induced moderate-to-high NAb titers to tier-1 viruses (e.g., vaccine strains and heterologous NAb-sensitive strains) but minimal NAb titers to tier-3 viruses that are most resistant to NAbs (e.g., NAb-resistant strains generally different from vaccine subtypes) (Table 3). FIV_Bang is placed under tier 2 because the majority of its expressed Env regions belong to subtype B and not to the vaccine subtypes A and D [18], and due to the low anti-FIV_Bang NAb titers induced by Fel-O-Vax^® FIV. Thus, our definition of tier-2 strains are heterologous strains from the same vaccine subtype or heterologous-subtype Env sequence, which are more resistant to Fel-O-Vax^® FIV than to prototype IWV.

3.6. Passive-transfer studies to determine the mechanism of prophylaxis and the FIV tiers

The role of vaccine-induced antibodies in the protection observed were further evaluated via passive-transfer studies using the antibodies generated from the Fel-O-Vax^® FIV-vaccinated cats before FIV_Pet or FIV_FC1 challenge (Table 4, Studies PT1, PT2, and PT3). In Study PT1, the pooled vaccine serum with anti-FIV_Pet NAb titer of 2000/mL conferred protection against FIV_Pet in all four recipients but not in controls immunized with saline or non-vaccine serum. All recipients of the pooled vaccine serum had NAb titers to FIV_Pet ranging from 50–1000 at 1 wpc (Study PT1, only average titers). All recipients of the vaccine serum, except for one, completely lost the anti-FIV_Pet NAbs by 24 wpc, while two control cats tested (other two untested) developed infection-induced anti-FIV_Pet NAb titers.

In Studies PT2 and PT3 (Table 4), passive transfer with purified vaccine-induced antibodies protected 7 of 8 recipients against FIV_Pet and 0 of 5 against FIV_FC1, while all 12 control cats became infected. In Study PT2, all recipients of vaccine antibodies had anti-FIV_Pet NAb titers ranging from 10–1000 at 1 wpc, but only one cat had an anti-FIV_FC1 NAb titer (50 NAb) at 1 wpc. This study supports the notion that the vaccine induced antibodies, but not other serum factors, were involved in the passive protection against the homologous tier-1 FIV_Pet. In Study PT3, the five recipients of the purified vaccine-induced antibodies had anti-FIV_Pet NAb titers ranging from 50–1000 at 1 wpc, while only two cats had anti-FIV_FC1 NAb titers of 10–50 at 1 wpc (Table 4, only average titers). Except for one, all unprotected cats as well as infected control cats had undetectable anti-FIV_FC1 NAb titers at 14 and 24 wpc. Thus, these results suggest that the homologous FIV_Pet is a tier-1 virus, while heterologous-subtype FIV_FC1 is a tier-3 virus.