Ad26.M.Env vaccination induced potent anti-ZIKV neutralizing antibodies and prior vaccination did not impact conception in female macaques.
Thirteen female macaques were immunized with 1011 vp of Ad26.M.Env expressing ZIKV M protein transmembrane domain lacking the peptide precursor (PR) and the envelope (Env) antigens (Ad26.M.Env) and were returned to the breeding colony 17 days post-vaccination (Fig. 1a). Dams were vaccinated a minimum of 2 months and a maximum of 7 months prior to challenge. An additional 13 females were selected that received sham vaccination that were placed in the breeding colony at the same time as the vaccine group. No adverse events were noted by clinical staff immediately following vaccination. In the weeks following vaccination, some animals had reports of weight loss, diarrhea, and trauma, but these clinical observations did not correlate temporarily with vaccine administration. A peak in total white blood cells was seen in vaccinated animals at four weeks post-vaccination likely due to vaccine immune stimulation (Suppl Fig. 1).
Females in all groups were monitored bi-weekly by ultrasound for pregnancy. Pregnant females in the Ad26.M.Env vaccine and sham groups were infected with 1x106 vp of ZIKV via the subcutaneous route ZIKV 6 weeks post-conception (10–30 weeks post-vaccination based on timing of confirmed pregnancy, Suppl. Table 1). Three pregnant sham animals were not infected to serve as normal pregnant controls. Plasma, sera, cerebral spinal fluid, urine, colorectal, cervical, and saliva samples were collected during pregnancy from all dams as indicated (Fig. 1a).
Neutralizing antibody titers were determined by both Immunospot focus reduction neutralization (FRNT) and by microneutralization assay (MN50 VNA). Before immunization, animals (OBF, 079 and 437) showed very low neutralization titers whereas the other six animals showed no neutralization titers when assayed with FRNT (Fig. 1b). Four weeks after immunization, all animals developed a neutralizing response that was maintained or only marginally decreased 8 weeks after immunization and in pre-challenge serum which was obtained 10 to 30 weeks after immunization (Fig. 1b,d). Four weeks after challenge, all animals of the Ad26.M.Env immunized group had increased ZIKV neutralization titers (Fig. 1b). Serum of animals of the non-immunized control group were assayed pre- and post- challenge. Pre-challenge, no neutralization titers were detected with FRNT analysis, whereas ZIKV neutralization titers developed after challenge (Fig. 1c). These FRNT results were confirmed by the MN50 neutralization assay (Suppl. Figure 2). Post-challenge neutralizing titers were comparable between Ad26.M.Env and sham vaccinated animals, consistent with minimal amnestic antibody responses in vaccinated animals (Fig. 1b, c, Suppl. Figure 2). Next, antibody responses against ZIKV NS1 protein were measured by ELISA. Ad26.M.Env does not contain a NS1 antigen. In accordance, Ad26.M.Env vaccinated or sham vaccinated animals had low (079) or undetectable NS1 binding antibody responses in pre-challenge samples. Four weeks after challenge, all 5 non-immunized dams developed high NS1-specific antibody titers (mean titer of 3.69 log10). Eight out of 9 dams that received Ad26.M.Env also developed NS1-specific titers after challenge although the group mean NS1 titer (1.75log10) was approximately 100-fold (2 log10) lower compared to the group mean NS1-titer in the non-immunized animals (Suppl. Figure 3).
Ad26.M.Env vaccination induced anti-Env cellular immune responses in macaques.
Env and prM directed cellular immune responses were measured by IFNγ ELISPOT on frozen PBMC’s isolated pre- immunization, post-immunization, pre-challenge, and post-challenge. Ad26.M.Env vaccination resulted in induction of ZIKV specific cellular responses (Fig. 2). The geometric mean Env-specific cellular immune responses in the group that received Ad26.M.Env was above 50 SFU per 106 PBMCs at week 4 and 8 after immunization, and at the pre-challenge timepoint (geomean SFU 55.03, 55.92, and 97.41, respectively). The Env-specific cellular immune responses after immunization were higher when compared to the Env-specific cellular immune responses pre-immunization, or in non-immunized animals which were both below the limit of detection (Fig. 2a,b). The prM-specific cellular responses were generally low and geometric mean responses do not exceed the cut-off of 50 SFU per 106 PBMCs (Fig. 2c,d). Notably, Env and prM cellular responses did not increase after challenge as compared to the pre-challenge timepoint indicating a lack of amnestic cellular responses in Ad26.M.Env vaccinated animals (Fig. 2a,c).
Ad26.M.Env vaccinated pregnant females were completely protected against ZIKV viremia in blood and tissues.
Pregnant females in the vaccine and sham groups were infected with 1x103 PFU Zika virus from the 2015 Brazilian epidemic at 6 weeks post-conception (being 10 to 30 weeks post vaccination). Vaccinated dams had no detectable virus in plasma post-ZIKV challenge even though animals were challenged from 10 to 30 weeks after vaccination, depending on the timepoint of conceiving (Fig. 3a). In contrast, sham vaccinated pregnant macaques all had detectable viral load, with a mean peak viremia of 5.5 log10 on day 7 post-challenge (Fig. 3b) consistent with peak viremia reported for ZIKV infected non-pregnant and pregnant macaques (22,32,33,36). On average, sham vaccinated animals had detectable virus for 42 days with a range of 7 to 56 days consistent with previous reports that pregnancy prolongs ZIKV viremia in rhesus monkeys (32,36). All colorectal, vaginal, saliva, and amniocentesis samples were negative for ZIKV for all ZIKV-challenged dams irrespective of vaccination status.
After confirmation of pregnancy, dams were monitored bi-weekly for fetal biometric analyses including measurements of biparietal diameter, occipitofrontal length, head circumference, and femur length by ultrasonography. No abnormalities were noted in fetal biometric parameters between study groups (Suppl. Figure 4) and fetal brain weights and brain:fetal body weight ratios at necropsy were similar across groups (Suppl. Figure 5).
Dams had scheduled Cesarian sections and euthanasia when fetuses were term, approximately 2 weeks prior to estimated delivery date. Maternal tissues previously shown to have detectable virus throughout pregnancy were collected for evaluation by RT-PCR for ZIKV viral RNA (vRNA). None of the nine Ad26.M.Env vaccinated dams had detectable vRNA in any tissues surveyed (Fig. 3c). All sham vaccinated dams had detectable vRNA in at least one of the analyzed tissues. 4/5 animals showed positive vRNA in maternal spleen, consistent with previous reports(23,32,34), and one dam had vRNA detected in the axillary LN. One dam had detectable vRNA in the uterus, and 3/5 dams had virus detectable in the placenta (Fig. 3d). Placenta pathology was evaluated for dams in all groups and evaluated by both a veterinary pathologist and a human gynecological pathologist specializing in placental histopathology. Histopathological placental findings in all groups were typical of near-term/term placentas in macaques with evidence of maternal thrombosis and infarction in all groups (Table 1)(41). Fetal: placental ratios were within the expected limits for term fetuses and did not vary significantly between groups (Suppl. Figure 5).
Table 1
Summary of gross and histopathological findings in dam and neonate tissues.
Animal ID
|
Condition
|
Group
|
Sex
|
Gross Findings
|
Histopathology- Fetus
|
Histopathology-Placenta
|
78
|
Control
|
3
|
F
|
NSF
|
Hemorrhage in lateral ventricle
|
moderate calcifications
|
583
|
Control
|
3
|
M
|
NSF
|
Cortical microcalcification
|
NSF
|
541
|
Control
|
3
|
M
|
NSF
|
Increased meningeal cellularity
|
NSF
|
05K
|
ZIKV
|
2
|
F
|
Hard, white lesion noted in liver
|
Cortical/neuroprogenitor dysplasia (mild); focal gliosis
|
placental thinning
|
560
|
ZIKV
|
2
|
F
|
absent occipital gyrus (L); focal proliferation of neuropil on the right cerebellar lateral hemisphere
|
Cortical/neuroprogenitor dysplasia (mild); Cortical and periventricular microcalcification; multifocal microhemorrhage; mild neuropil vacuolation/rarefication; multifocal meningeal proliferation; increased meningeal cellularity
|
NSF
|
05L
|
ZIKV
|
2
|
M
|
Dilated lateral ventricle upon examination of fixed specimens; asymmetry L parietal lobe
|
Cortical microcalcification
|
thrombosis maternal vessel
|
02X
|
ZIKV
|
2
|
M
|
Absent gyrus in parietal cortex; cloudy CSF
|
Microcalcification within neuroprogenitor clusters, perivascular edema, and necrosis; mild neuropil vacuolation/rarefication; focal gliosis
|
NSF
|
558
|
ZIKV
|
2
|
M
|
Enlarged ventricle noted upon examination of fixed specimens
|
Cortical/neuroprogenitor dysplasia (mild); cortical microcalcification; hemorrhage in lateral ventricle; multifocal gliosis; increased meningeal cellularity; multifocal spinal cord microhemorrhage
|
Moderate infarction/necrosis chorionic plate; thrombosis maternal vessel
|
07G
|
Vaccinated, ZIKV
|
1
|
M
|
NSF
|
Cortical/neuroprogenitor dysplasia (mild); mild neuropil vacuolation/rarefication; focal gliosis
|
thrombosis maternal vessel
|
0BF
|
Vaccinated, ZIKV
|
1
|
M
|
NSF
|
NSF
|
Mild abruption; thrombosis maternal vessel
|
437
|
Vaccinated, ZIKV
|
1
|
M
|
NSF
|
NSF
|
Mild infarction/necrosis chorionic plate; placental thinning; thrombosis maternal vessel
|
07M
|
Vaccinated, ZIKV
|
1
|
F
|
NSF
|
Cortical microcalcification; multifocal microhemorrhage; mild neuropil vacuolation/rarefication
|
NSF
|
OC1
|
Vaccinated, ZIKV
|
1
|
F
|
NSF
|
NSF
|
NSF
|
OBE
|
Vaccinated, ZIKV
|
1
|
F
|
NSF
|
NSF
|
Not evaluated; Vaginal delivery infant
|
79
|
Vaccinated, ZIKV
|
1
|
F
|
NSF
|
NSF
|
NSF
|
32
|
Vaccinated, ZIKV
|
1
|
F
|
NSF
|
Mild neuropil vacuolation/rarefication
|
Mild infarction/necrosis chorionic plate; thrombosis maternal vessels
|
Neonates born to Ad26.M.Env vaccinated dams were negative for ZIKV virus and had no ZIKV-associated histopathological abnormalities.
Following Cesarian section and euthanasia, fetuses were inspected for gross abnormalities and fetal tissues collected for histopathology and evaluation of vRNA. Fetuses of vaccinated dams had no evidence of viral replication in tissues (Fig. 4a, upper) while 2/5 fetuses from sham vaccinated dams had detectable virus in tissues, one of which (Fetus 560) had extensive detection of ZIKV in the brain (Fig. 4a, lower). Histopathologic evaluation of brain from fetuses born to Ad26.M.Env vaccinated dams showed no evidence of previously reported ZIKV neuropathology including microcalcifications and perivascular edema (Fig. 4b-e, Fig. 5). Sham vaccinated fetuses had a constellation of abnormal findings (Table 1) including a gross cerebellar malformation (Fig. 4f), asymmetry of the left parietal lobe (Suppl. Figure 6, Table 1), and a gross dystrophic calcification on the liver (Table 1), focal edema (Fig. 4g), microcalcification (Fig. 4h), and meningeal proliferation (Fig. 4i) However, overall the sham group had fewer histopathological findings than previously reported (Fig. 5)(32) and gross and histological abnormalities in sham vaccinated, challenged fetuses could not be definitively linked to ZIKV viral replication.