Considering the importance of adherence in the effectiveness of an anti-HIV microbicide strategy,1
we think that the most efficacious products will be those that remain effective when used under diverse regimens according to the unique needs and desires of the users. Problems with adherence in the CAPRISA 004 trial (1% tenofovir gel) suggest that without user flexibility for dosing, gel may not be effective over the long term. Even though macaque studies are starting to address this by varying the timing of the tenofovir gel relative to virus in a repeat challenge model,16,17
more diverse regimens with additional and alternative APIs are needed. Building on previous data showing that repeated vaginal use of MIV-150/ZA/CG and ZA/CG gels reduced vaginal SHIV-RT acquisition in macaques,10
we evaluated the vaginal protection offered by a single application of these gels. Since vaginal gels might be used rectally and an ideal product could be used to prevent both vaginal and rectal HIV infection, we also obtained efficacy data on rectal application of the MIV-150/ZA/CG against rectal SHIV-RT infection.
Vaginal application of the MIV-150/ZA/CG gel was significantly protective compared to CG when applied once 24
h before challenge or both 24
h before and 1
h after challenge. Protection was the same as we previously observed for repeated application (1/14 infected for single vs. 2/28 infected for repeated10
), indicating that the MIV-150/ZA/CG gel is as effective when used once prior to virus exposure as when used regularly over a 2- to 4-week period. Although a single vaginal application of the ZA/CG gel 8–24
h before challenge was not significantly protective compared to the CG gel, the effect of this regimen was also not statistically different from the significant protection we previously observed for repeated application of this gel (6/14 infected for single vs. 4/21 infected for repeated,10
=0.16). Had we been able to test the ZA/CG gel in a larger group of animals, we might have been able to demonstrate a modest effect of a single dose. Nevertheless, our data clearly demonstrate that repeated application is needed for greater efficacy by the ZA/CG gel.
The benefit of repeated dosing for the ZA/CG gel that is not required for the MIV-150/ZA/CG gel may lie in the mechanism of action of ZA. Building on earlier reports,9,18,19
we have demonstrated that ZA has antiviral activity in vitro
(Fernández-Romero, Hsu, and Robbiani, unpublished). A recent study suggests that this is at least partially mediated by zinc's ability to impede RT activity.20
, it is possible that ZA renders the mucosal surface more resistant to infection, and repeated application may provide positive feedback to promote or sustain this antiviral state.18,19,21,22
Additionally, the amount of zinc absorbed into the tissues may be greater with repeated application of the ZA/CG gel, and this may translate into greater efficacy (through antiviral and/or immunomodulatory mechanisms).
Importantly, limited protection was observed when the gels were applied only 1
h post challenge. This demonstrates that the gels are unsuitable for post exposure prophylaxis in a single dose and highlights that in the animals given the MIV-150/ZA/CG gel both 24
h pre-challenge and 1
h post challenge, the 24
h preexposure dose was the protective component. Preliminary data in an activated T cell system suggest greater in vitro
antiviral activity of ZA after repeated dosing post virus exposure compared to a single co-exposure to ZA and virus (Fernández-Romero, unpublished). However, whether repeated dosing post virus exposure is effective in vivo
remains to be determined.
We previously observed that a single rectal dose of the MIV-150/CG gel (which was only 56% protective vaginally compared with the 89% protection seen with the MIV-150/ZA/CG gel) completely protected macaques (zero of four infected, 0%) when given 4
h before rectal SHIV-RT challenge.11
Since vaginal gels will likely be used rectally, we wanted to establish the duration of protection afforded by the more potent MIV-150/ZA/CG gel when applied rectally (using the time points tested for vaginal challenge). However, we found that the MIV-150/ZA/CG gel had limited efficacy rectally when used 8–24
h before challenge, only slightly (and not significantly) reducing infection compared to the CG gel. Since the original rectal testing of the MIV-150/CG gel did not include a CG gel only group, we cannot be certain that there was no barrier effect of CG (within the MIV-150/CG gel) at the 4
h time point that contributed to the protection seen.11
Notably, there is basically no barrier effect of CG when given 8
h prior to challenge (compare the five/six infected in the CG group herein to the four/four infected in the methyl cellulose placebos in our earlier study11
However, independently these data underscore that this gel can prevent rectal infection if used closer to the time of challenge (even if this might be contributed to by the CG barrier effect). In practice, rectal gel use is more likely to coincide with the timing of sex than a vaginal gel since a lubricant is often used to facilitate anal intercourse.23
Thus, the real world need for rectal gels that offer several hours of protection is limited. Moreover, knowing that a vaginal gel does not exacerbate rectal infection is important. Despite in vitro
data suggesting a potential enhancing effect of CG on HIV infection,4,24,25
we have found no evidence of CG-mediated enhancement in macaques following vaginal or rectal application, even when challenging up to 24
h after gel dosing (data herein and unpublished observations).4,10,11,26
This also supports the earlier findings from the Phase 3 trial testing the efficacy of CG against HIV infection, where the HIV infection frequency was reduced, albeit not significantly, in the CG arm27
and that HIV shedding was not enhanced in HIV-infected women using CG.28
Although the MIV-150/ZA/CG gel had greater activity vaginally than the gels containing either only MIV-150 or ZA, specific controls of animals treated rectally with the MIV-150/CG or ZA/CG gels would be needed to verify the contribution of both agents rectally. It is possible that ZA may have more limited activity against rectal than vaginal SHIV-RT infection, which would lead the MIV-150/ZA/CG gel to perform much like the MIV-150/CG gel. However, the ZA/CG gel has been shown to effectively prevent high-dose rectal HSV-2 infection in mice,9
indicating that ZA is active in the presence of rectal enzymes and microflora.
Twelve of 13 animals that became infected after treatment with the MIV-150/ZA/CG gel (vaginally or rectally) had SHIV-RT carrying the wild-type RT gene. In one clone (of 17) from animal GD42 we detected the V179G mutation in the RT gene. V179 is a highly polymorphic position. V179F has been described as an important mutation frequently selected by etravirine, decreasing susceptibility to this drug when it is present in combination with Y181C/I/N.29
However, V179D/E are often found in treatment-naive individuals, with no substantial impact on NNRTI-containing treatment when present by themselves.29
A recent review noted that the prevalence of V179G in NNRTI-resistant vs. non-NNRTI-resistant samples is 0.15 vs. 0.03%, respectively, and that it resulted in an IC50
fold change of only 0.6 for efavirenz and etravirine.30
The effect of this mutation on MIV-150 susceptibility still needs to be tested phenotypically, but because of the polymorphism of this amino acid position, it is unlikely that this mutation will have a profound effect on MIV-150 antiviral activity. Finding predominantly wild-type virus (especially in the plasma) is not surprising since MIV-150 was not detected in the plasma after 24
pg/ml), indicating that any MIV-150 absorbed into the blood was cleared rapidly (as we have seen in rats treated with the MIV-150/ZA/CG gel; Fernández-Romero and Zydowsky, unpublished). We were unable to perform pharmacokinetic studies to measure MIV-150 levels in the tissues at the time of challenge and so cannot rule out that there would have been differences between the various tissue drug levels (or earlier plasma levels) that might explain the differences in efficacy (vaginally vs. rectally). Additionally, the emergence of drug-resistant mutations in virus within the tissues (where virus might be exposed to drug over a longer period of time) is being investigated using SHIV-RT-infected macaques.
Expanding our original findings (i.e., repeated use of the MIV-150/ZA/CG gel prevented vaginal immunodeficiency virus infection), we provide evidence suggesting that even sporadic use of the MIV-150/ZA/CG gel blocks vaginal immunodeficiency virus and HSV-2 infections. Future work will address the window of protection for rectal use. Our findings encourage the advancement of this product for clinical testing.