key: cord-0429260-l9x3f9e5
authors: Kilgore, Paul B.; Sha, Jian; Hendrix, Emily K.; Motin, Vladimir L.; Chopra, Ashok K.
title: Combinatorial viral vector-based and live-attenuated vaccines without an adjuvant to generate broader immune responses to effectively combat pneumonic plague
date: 2021-10-29
journal: bioRxiv
DOI: 10.1101/2021.10.28.466384
sha: 1d4d2cf000a6887003f77631174e0dff7bdb3a1f
doc_id: 429260
cord_uid: l9x3f9e5

Mice immunized with a combination of an adenovirus vector (Ad5-YFV) and live-attenuated (LMA)-based vaccines were evaluated for protective efficacy against pneumonic plague. While the Ad5-YFV vaccine harbors a fusion cassette of three genes encoding YscF, F1, and LcrV, LMA represents a mutant of parental Yersinia pestis CO92 deleted for genes encoding Lpp, MsbB, and Ail. Ad5-YFV and LMA were either administered simultaneously (1-dose regimen) or 21 days apart in various order and route of administration combinations (2-dose regimen). The 2-dose regimen induced robust immune responses to provide full protection to animals against parental CO92 and its isogenic F1 (CAF-)-deletion mutant challenges during both short- and long-term studies. Mice intranasally (i.n.) immunized with Ad5-YFV first followed by LMA (i.n. or intramuscularly [i.m.]) had higher T- and B-cell proliferative responses and LcrV antibody titers than those in mice vaccinated with LMA (i.n. or i.m.) first ahead of Ad5-YFV (i.n.) during the long-term study. Specifically, the needle- and adjuvant-free vaccine combination (i.n.) is ideal for use in plague endemic regions. Conversely, with a 1-dose regimen, mice vaccinated with Ad5-YFV i.n. and LMA by the i.m. route provided complete protection to animals against CO92 and its CAF- mutant challenges, and elicited Th1/Th2, as well as Th17 responses, thus suitable for emergency vaccination during a plague outbreak or bioterrorist attack. This is a first study in which a viral vector-based and live-attenuated vaccines were effectively used in combination representing adjuvant- and/or needle- free immunization, with each vaccine triggering a distinct cellular immune response. Importance Yersinia pestis, the causative agent of plague, is a Tier-1 select agent and a re-emerging human pathogen. A 2017 outbreak in Madagascar with >75% of cases being pneumonic and 8.6% causalities emphasized the importance of the disease. The World Health Organization has indicated an urgent need to develop new generation subunit and live-attenuated plague vaccines. We have developed a subunit vaccine including three components (YscF, F1, and LcrV) using an adenovirus platform (Ad5-YFV). In addition, we have deleted virulence genes of Y. pestis (e.g., lpp, msbB, and ail) to develop a live-attenuated vaccine (LMA). Both of these vaccines generated robust humoral and cellular immunity and were highly efficacious in several animal models. We hypothesized the use of a heterologous prime-boost strategy or administrating both vaccines simultaneously could provide an adjuvant- and/or a needle- free vaccine(s) which have attributes of both vaccines for use in endemic regions and during an emergency situation.

the Th1 immune response was favored after vaccination of mice with the Ad5-YFV vaccine over 100 Th2, possibly due to the Ad5 vector used, while the Th2 immune response was favored after 101 immunization of animals with the LMA vaccine (26, 28) . In our past studies, the Ad5-YFV 102 vaccine was always delivered intranasally (i.n.), while the LMA vaccine was administered by the 103 i.m. or the i.n. route (19, (25) (26) (27) (28) . 104 Like other live-attenuated vaccines, one of the major advantages of LMA is that it is the safety profile, especially in immunocompromised individuals, and the potential concern for 111 reversion (35-37). However, the LMA mutant was rationally designed with complete deletion of 112 three genes located at different locations on the bacterial genome (19). Because of its high level 113 of virulence attenuation and rapid clearance (within 12-24 h) while retaining immunogenicity in 114 animals (27) , LMA vaccine was excluded from the Centers for Disease Control and Prevention 115 (CDC) select agent list (https://www.selectagents.gov/sat/exclusions/hhs.html).

The Ad5-YFV vaccine only incorporates three plague antigens: F1, LcrV and YscF, and 117 the F1-minus strains of Y. pestis have been isolated from humans/animals and are as virulent as 118 the wild-type (WT) plague bacterium (38, 39). In addition, hypervariable regions within the 119 LcrV protein have been described and antibody responses to these LcrV variants are not cross-120 protective (18). Therefore, we have successfully added a third protective antigen YscF in this 121 vaccine to circumvent disadvantages of F1-V-based vaccines (25, 40). However, it is still 122 plausible that the Ad5-YFV vaccine alone may not be efficacious against all circulating Y. pestis 123 variants, although it is shown to be protective (100%) against CAFmutant of CO92 (26). 124 Importantly, both of our vaccines do not require an adjuvant to boost immune responses, unlike 125 F1-V-based vaccines which employ Alum (8, 9, 11, (41) (42) (43) . 126 To alleviate some of the above concerns, in this study, we carried out a heterologous 127 immunization strategy in which both LMA and Ad5-YFV vaccines were administered either 

Virulence and immunogenic characterization of the LMA vaccine candidate in iron 136 over loaded conventional and or in immunocompromised mice 137 We have previously shown LMA mutant to be highly attenuated in conventional 138 (immunocompetent) mice (19, 27) . To further evaluate its attenuation, we tested safety of the 139 LMA mutant during iron overload conditions in conventional mice to mimic hemochromatosis. 140 We also tested its safety in immunocompromised Rag1 KO mice. 141 We demonstrated that all of the mice challenged with the LMA vaccine (5x10 6 CFU, 142 more than double the vaccination dose) by the i.n. route survived irrespective of whether the 143 animals were iron-overloaded or not (Fig. 1A) with a minimal loss in the body weight (Fig. 1B) .

On the contrary, mice infected with the same dose of the KIM/D27 strain died by day 5 in the 145 presence of FeCl 2 , while 80% of the animals succumbed to infection without iron-overload ( Fig.   146   1A) . At a lower challenge dose of 2x10 5 CFU, 80% of the iron-overloaded mice succumbed by 147 day 7 while only 20% of the non-iron-overloaded mice succumbed (Fig. 1A) . In contrast to the 148 LMA mutant, KIM/D27 strain-infected mice showed a dramatic loss in the body weight even at 149 the lower challenge dose of 2x10 5 CFU (Fig. 1B) . 150 We then infected Rag1 KO mice with 4 LD 50 of CO92 by either the i.n. or the i.m. route 151 since these two routes were previously used for vaccination studies with LMA (19, 27, 28). As 152 shown in Fig. S1A , 100% of mice infected by the i.n. or the i.m. route succumbed to infection by 153 day 4-5 p.i., with up to ~20% body weight loss by day 4. We then examined bacterial burden in 154 the lungs, liver, and spleen of these mice and found greater than 10 8 CFU of Y. pestis in both the 155 lungs and the spleen, while a significantly higher Y. pestis burden, with up to a more than 10 9 156 CFU, was observed in the liver (Fig. S1B) . 157 We then evaluated the LMA mutant, when delivered at a vaccination dose of 2.0 x 10 6 158 CFU by both i.n. and i.m. routes, in Rag1 KO mice. Five days p.i., five mice infected with LMA 159 from each infection route were necropsied and examined for the presence of the mutant either at 160 the infection site (lungs or muscle) or in the spleen as the result of systematic dissemination. As 161 shown in Fig. 2A , there was no detectable LMA mutant in the spleen of mice infected by either 162 the i.n. or the i.m. route. We also did not detect any LMA mutant at the injection site of muscle. 163 We did enumerate 300 CFU in the lungs of one mouse that was infected by the i.n. route; 164 however, this number was much lower than the initial infection dose of 2.0 x 10 6 CFU. Further, 165 no LMA mutant was detected in the lungs of other mice infected by the i.n. route ( Fig. 2A) . This 166 was also indicated by the fact that all the mice infected with 2.0 x 10 6 CFU of LMA survived up 167 to 28 days p.i. (Fig. 2B) without any clinical signs of the disease and a minimal body weight loss 168 similar to that shown in conventional mice (Fig. 1B) . 169 These surviving mice were then challenged with 6 LD 50 of CO92. As expected, all LMA 170 infected Rag1 KO mice succumbed to CO92 challenge with an overall >10 8 CFU of Y. pestis 171 CO92 present in the lungs (Fig. 2C) with ~15% loss in body weight over time. Further, there 172 were no F1-V specific IgG antibodies in the sera of LMA (pooled from i.n. and i.m. infected) 173 Rag1 KO mice on day 3 post CO92 challenge as compared to naïve Rag1 KO mice. However, a 174 significantly higher level of F1-V IgG antibodies was generated in the LMA-immunized 175 conventional (immunocompetent) mice (pooled sera from i.n. and i.m. infected) from a parallel 176 independent study (Fig. 2D) .

Strong immune responses were elicited in conventional mice by heterologous vaccination 178 with either a 1-or 2-dose (prime-boost) regimen 179 Mice were immunized with Ad5-YFV and LMA vaccines in either a 1-or 2-dose 180 regimen. In a 1-dose regimen, both Ad5-YFV and LMA vaccines were administered 181 simultaneously, while in a 2-dose regimen, Ad5-YFV and LMA vaccines were delivered 21 days 182 apart in various order and route combinations (Fig. 3A) . The immunization schedule for either 1-183 or 2-dose regimens is depicted in Fig. 3B ). Three weeks after completion of the vaccinations, 184 the immunized and control mice were challenged with 100 LD 50 of either CO92 or its F1 185 deletion mutant CAF -. As shown in Fig. 3C and D, all of 2-dose heterologous prime-boost 186 vaccinated mice, regardless of the order in which the vaccines were administered or the route of 187 vaccination by which LMA was delivered, were 100% protected from both CO92 and its CAF -188 strain challenge with no body weight loss and other clinical symptoms of the disease. A 100% 189 protection was also observed for mice simultaneously immunized with Ad5-YFV (i.n.) and LMA 190 (i.m) during CO92 and CAFchallenges. However, when both Ad5-YFV and LMA vaccines 191 were simultaneously administered i.n., the immunized mice had 50% (during CAFstrain 192 challenge) and 63% (during CO92 challenge) survival rates ( Fig. 3E and F) . 193 We then measured IgG antibody titers to rF1-V fusion protein in sera of immunized mice 194 collected on day 42 prior to the challenges. In general, all vaccinated groups of animals had 195 notable increases in antibody titers which were 2-3 logs higher compared to that of the naive 196 controls (Fig. 4A) . Among the immunized mice, relatively lower F1-V antibody titers were noted 197 in animals immunized with either a 2-dose regimen of Ad5-YFV (i.n.)-LMA (i.m) or a 1-dose 198 regimen of Ad5-YFV (i.n.) + LMA (i.n.). However, a significant difference was only observed 199 between the 2 simultaneously immunized groups of mice (Fig. 4A , red versus blue bars). 200 We also examined the isotypes of F1-V IgG antibodies to gauge Th1 versus Th2 bias. In (Fig. 4B) . In contrast, when the LMA vaccine was delivered as the first dose (crimson and 209 orange bars), higher levels of IgG1 over IgG2a were observed but again these differences were 210 only significant when LMA was administered by the i.m. route as shown in orange bars ( Fig.   211 4B). 212 We next examined cell-mediated immune responses in immunized mice from the groups 213 which showed 100% protection during the Y. pestis challenges (Fig. 3C-F) . In a separate 214 experiment, splenocytes were isolated from vaccinated mice 21 days after the last vaccination 215 dose and stimulated with PMA and Ionomycin. All vaccination groups had significantly higher 216 populations of CD4 + IFNγ + , CD8 + IFNγ + and CD4 + Il-17 + cells than those of naive mice (Fig 4C- 217 E). Among the vaccinated groups, mice i.n. immunized with either LMA or Ad5-YFV first in a 218 2-dose regimen (teal and crimson bars) showed slightly higher percentages of IFNγ producing 219 CD4 + and CD8 + T cells than all other groups of immunized mice; however, no significant 220 differences were observed ( Fig. 4C and D) . 221 On the other hand, notably higher population of IL-17 producing CD4 + T cell was 

As shown in Fig. 5C , a similar levels of F1-V specific antibodies were detected in the 239 sera across all vaccinated groups of mice and were all significantly higher (>2 logs) than that of 240 naïve control animals (Fig. 5C) . Further, antibody isotype analysis revealed a generally balanced 241 level of IgG1 and IgG2a in the immunized mice except for animals immunized first with Ad5-242 YFV (teal and purple bars) which had higher levels of IgG2a over IgG1. However, the difference 243 was only significant in mice immunized with Ad5-YFV (i.n.)-LMA (i.n.) (Fig. 5D , teal bars).

To further dissect the antibodies elicited by the vaccination, we evaluated antibody titers 245 to each individual plague antigen F1, LcrV, and YscF that were the components of Ad5-YFV controls. (Fig. 7D) . 279 We then examined sera of mice for IgA post vaccination and post challenge with CO92, 280 and no differences in titers were noted (Fig. S2) , suggesting infection did not further enhance 281 serum IgA levels. A similar study will be performed in the future assessing IgA levels in BALF 282 post vaccination and post challenge. Mice from the Ad5-YFV (i.n.)-LMA (i.n.) vaccinated group 283 had slightly lower level of IgA than the other heterologous prime-boost vaccinated groups of 284 animals, although this differences were not significant (Fig. 7D) . Although it is expected that in 285 pneumonic plague, IgA would be significantly contributing to host protection, some studies 286 indicated minimal protective role of IgA (31, 45). However, whether the immune status of the 287 host could be a contributing factor in IgA-associated protection is unclear and needs further 288 investigation.

To gauge cell-mediated immunity of vaccinated mice in response to CO92 challenge, on 290 day 3 p.i., splenocytes were isolated from both immunized and control animals and subjected to 291 flow analysis after PMA/Ionomycin stimulation. We noted statistically higher IFNγ producing 292 CD4 + and CD8 + T-cell populations across all of the vaccinated mice as compared to that of 293 naïve control animals ( Fig. 8A and B) , and a similar trend was observed for the IL-17 producing 294 CD4 + T-cells (Fig. 8C) . 295 More specifically, a significantly higher level of CD4 + IFNγ + population was observed (Fig. 8B) . 301 In terms of CD4 + IL-17 + population, mice immunized first with LMA either by the i.n. or 302 the i.m. route generally revealed better levels than mice immunized with Ad5-YFV vaccine first. at the comparable levels (IL-1α and IL-1β) to those of naïve control mice (Fig. 9) . However, in 317 response to Y. pestis infection, significantly increased proinflammatory cytokines were observed 318 only in naïve control mice, while the levels of these cytokines were low and almost unchanged in 319 vaccinated animals (Fig. 9) . For the anti-proinflammatory cytokine IL-10, a significant increase 320 was generally observed in all of the immunized mice as compared to that of naïve controls at the challenge time point (Fig. 9) .

In contrast to proinflammatory cytokines, the Th1-and Th2-related cytokines such as IL-327 2, IL-12(p70), IFNγ, IL-4, IL-5, and IL-13 were generally increased in all immunized mice as 328 compared to that of naïve controls at the post-immunization time point (Fig. 10) In analysis of chemokine production, the overall level of chemokines in all immunized 347 mice were low and comparable with their corresponding naïve controls at the post immunization 348 time point except the GM-CSF which was significantly elevated (Fig. 11) . In response to Y. pestis infection, the CXCL1, RANTES, and G-CSF behaved similarly to proinflammatory 350 chemokines and were significantly increased only in naïve control mice but were at low levels 351 and largely unchanged in all immunized and challenged animals (Fig. 11) . In contrast, CCL2, (Fig. 11) . 

Unlike KIM/D27 strain which exhibited more virulence under an iron-overload 372 environment, LMA vaccine was not affected by the presence of more iron in mice (Fig. 1) . The 373 Rag1 gene defect in humans is associated with a broad spectrum of clinical and immunological 374 phenotypes, and is one of the major causes of human immune deficiency (PID) (49, 50) . The

Rag1 KO mice receiving up to 2.0 x 10 6 CFU of LMA, which is equivalent to 20,000 LD 50 of 376 CO92, did not exhibit any clinical symptoms of the disease and the LMA mutant rapidly cleared 377 from these mice (Fig. 2) . These data demonstrated a high degree of attenuation imparted by the 378 selected mutations in LMA and provided indication that the vaccine would be suitable for use in 379 immunocompromised individuals. 380 We then implemented a heterologous immunization strategy in which both LMA and exception was the group of mice that received Ad5-YFV and LMA vaccines simultaneously by 397 the i.n. route and had 50 to 63% survival rates during CO92 or its CAFstrain challenges (Fig. 3) .

This relatively lesser protection rate in mice was correlated well with lower F1-V specific More specifically, when the Ad5-YFV vaccine was delivered first, Th1 immune response 421 was more pronounced based on F1-V specific IgG2a/IgG1 antibody ratio (Fig. 4B ) and the 422 splenic cytokine profiles (Fig. 10) . Likewise, when LMA vaccine was administered first 423 followed by that of Ad5-YFV, the Th2 immune response was favored ( Fig. 3B and Fig. 8C ) 424 along with that of Th17 ( Fig. 8C and Fig. 10 ) response. Further, clear differences were noted in 425 terms of T-and B-cell proliferation when mice were immunized first with the Ad5-YFV vaccine 426 in response to stimulation with rF1-V and had higher LcrV antibody titers when compared to 427 animals receiving LMA vaccine first during the long-term study ( Fig. 5E and Fig. 6 ). Therefore, 428 in this regard, delivering the Ad5-YFV vaccine first followed by that of LMA vaccine would be 429 preferred, and is much more attractive based on safety and that both vaccines can be 430 administered i.n., thus developing a more acceptable adjuvant-and needle-free administration 431 protocol from the public health prospective.

The heterologous vaccination strategies have been previously used for diseases in which Our future studies will address three important questions and include: 1) 505 comprehensively assessing potency of memory responses (T-and B-cells) that would navigate 506 us on dosing strategies; 2) assessing antibody potency in neutralizing Y. pestis infection, which 507 would also support our heterologous prime-boost approach with two vaccines and potentially 508 could reveal other additional differences that are important in humans; and 3) testing 509 heterologous prime-boost strategy in humanized mouse model and non-human primates to gauge 510 superiority of our approach compared to competing vaccine candidates, and that our Ad5-YFV 511 and LMA combination would be highly efficacious in humans. Splenocytes were then seeded into 24 well tissue culture plates at a density of 2.0 x 10 6 615 cells/well. Four wells/mouse/plate were treated with ionomycin (750 ng/mL, calcium ionophore), PMA (phorbol 12-myristate 13-acetate, protein kinase C activator, 50 ng/mL), and Brefeldin A 617 (5 µg/mL) for 5 h at 37° C in a 5% CO 2 incubator. Stimulation of splenocytes with PMA and 618 ionomycin leads to activation of several intracellular signaling pathways, bypassing the T cell 

The splenocytes were then subjected to flow cytometry, and data analyzed as we previously 640 described (28) . The percent of BrdU positive cells in CD3 and CD19 positive populations were 641 calculated using FACSDiva software.

To assess cytokine production, cell supernatants were collected from a duplicate plate 643 above after stimulation with rF1-V (100 µg/ml) for 72 h at 37°C. For these studies, we used Y. immunized and naïve control mice as described in Fig. 3 . ELISA was performed to evaluate 965 specific F1-V total IgG titers (A) as well as its isotype IgG1 and IgG2a titers (B) . In a separate 966 experiment, mice (n=5) were similarly immunized as described in Fig. 3 , however, the group in

Yersinia pestis--etiologic agent of plague

Human plague: An old scourge that needs new answers

Using evidence to inform response to the 2017 plague outbreak in Madagascar: A 671 view from the WHO African Regional Office

Epidemiological characteristics of an urban plague epidemic in Madagascar

The 2017 plague outbreak in 680 Madagascar: Data descriptions and epidemic modelling

Pneumonic Plague: The 683

Darker Side of Yersinia pestis

Plague vaccines: new developments in 685 an ongoing search

A phase I safety and immunogenicity dose escalation trial of plague 689 vaccine, Flagellin/F1/V, in healthy adult volunteers (DMID 08-0066)

One 692 year immunogenicity and safety of subunit plague vaccine in Chinese healthy adults: An 693 extended open-label study

Immunogenicity and safety of subunit plague vaccine: A randomized 696 phase 2a clinical trial

Protection against experimental bubonic and pneumonic plague by 699 a recombinant capsular F1-V antigen fusion protein vaccine

Recombinant V antigen protects mice against pneumonic and 702 bubonic plague caused by F1-capsule-positive and -negative strains of Yersinia pestis

Impact of Toll-Like Receptor-Specific 707

Agonists on the Host Immune Response to the Yersinia pestis Plague rF1V Vaccine. Front

Development of 710 in vitro correlate assays of immunity to infection with Yersinia pestis

The difference in the IcrV sequences between Y. pestis and Y. 714 pseudotuberculosis and its application for characterization of Y. pseudotuberculosis 715 strains

Passive immunity to yersiniae 717 mediated by anti-recombinant V antigen and protein A-V antigen fusion peptide

Amino acid and structural variability of 721

Yersinia pestis LcrV protein

Polymorphism in the Yersinia LcrV antigen enables immune escape from the protection 725 conferred by an LcrV-secreting Lactococcus lactis in a pseudotuberculosis mouse model. 726 Front Immunol 10

Combinational Deletion of 729

Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while 730

Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

Cell activation and apoptosis by bacterial lipoproteins 734 through Toll-like receptor-2

Braun lipoprotein 737 (Lpp) contributes to virulence of Yersiniae: Potential role of Lpp in inducing bubonic and 738 pneumonic plague

Characterization of late acyltransferase genes of Yersinia pestis and their role in 741 temperature-dependent lipid A variation

Deletion of the braun lipoprotein-encoding gene 744 and altering the function of lipopolysaccharide attenuate the plague bacterium

Resistance of Yersinia pestis to complement-dependent killing is mediated by the 748

Ail outer membrane protein

Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers 753

Complete Protection against Plague in Mice and Nonhuman Primates

A new generation needle-756 and adjuvant-free trivalent plague vaccine utilizing adenovirus-5 nanoparticle platform

Intramuscular Immunization of Mice with a Live-Attenuated Triple 760

Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To 761

Immunisation of 764 two rodent species with new live-attenuated mutants of Yersinia pestis CO92 induces 765 protective long-term humoral-and cell-mediated immunity against pneumonic plague

Defense against Pulmonary Yersinia pestis Infection

Protection elicited by attenuated live Yersinia pestis vaccine strains against lethal 772 infection with virulent Y. pestis

A recombinant attenuated Yersinia 774 pseudotuberculosis vaccine delivering a Y. pestis YopENt138-LcrV fusion elicits broad 775 protection against plague and yersiniosis in mice

Yersinia pestis and plague: an updated view on evolution, virulence determinants, immune 778 subversion, vaccination and diagnostics. Microbes Infect

Oral vaccination against plague using Yersinia 780 pseudotuberculosis

Humoral and cellular immune correlates of protection against bubonic plague by a 783 live Yersinia pseudotuberculosis vaccine

Vaccination of solid organ transplant candidates and 785 recipients: Guidelines from the American society of transplantation infectious diseases 786 community of practice

Risks associated with the use of live-788 attenuated vaccine poliovirus strains and the strategies for control and eradication of 789 paralytic poliomyelitis

Reversion of Cold-Adapted Live Attenuated 792 Influenza Vaccine into a Pathogenic Virus

F1-negative natural Y. pestis Strains

Characterization of an F1 deletion mutant of Yersinia pestis CO92, pathogenic role of F1 798 antigen in bubonic and pneumonic plague, and evaluation of sensitivity and specificity of 799 F1 antigen capture-based dipsticks

Immunization of mice with YscF 801 provides protection from Yersinia pestis infections

Long-term observation of subunit vaccine F1-rV270 against Yersinia pestis in mice

Short-and long-806 term efficacy of single-dose sub unit vaccines against Yersinia pestis in mice

Recombinant (F1 + V) vaccine protects cynomolgus macaques against 810 pneumonic plague

Accumulation of 812 bromodeoxyuridine-labeled cells in central and peripheral lymphoid organs: minimal 813 estimates of prodution and turnover rates of mature lymphocytes

Effective plague vaccination via oral delivery of plant cells expressing F1-V antigens in 817 chloroplasts

Efficacy trials of Plague vaccines: endpoints, trial design, site

A "prime-880 pull" vaccine strategy has a modest effect on local and systemic antibody responses to 881 HIV gp140 in mice

A Prime-Pull-Amplify Vaccination Strategy To 884

Maximize Induction of Circulating and Genital-Resident Intraepithelial CD8 + Memory T 885 Cells

A Systemic Prime-Intrarectal Pull Strategy Raises Rectum-888

Resident CD8+ T Cells for Effective Protection in a Murine Model of LM-OVA Infection

Front Immunol 11

A vaccine strategy that protects against genital herpes by 891 establishing local memory T cells

Tissue-resident memory T cells

Simultaneous Aerosol and Intramuscular Immunization with Influenza Vaccine Induces 896

Powerful Protective Local T Cell and Systemic Antibody Immune Responses in Pigs

Absence of inflammation and pneumonia during 899 infection with nonpigmented Yersinia pestis reveals a new role for the pgm locus in 900 pathogenesis

Braun lipoprotein 903 (Lpp) contributes to virulence of yersiniae: Potential role of Lpp in inducing bubonic and 904 pneumonic plague

In vivo thymocyte maturation. BUdR labeling of cycling thymocytes and 906 phenotypic analysis of their progeny support the single lineage model

and p120 expression by flow cytometric BrdUrd-Hoechst/7AAD and immunolabeling 910 technique

which mice were simultaneously immunized with LMA and Ad5-YFV i.n. was excluded. 968 Twenty-one days after completion of the vaccination course, spleens were harvested. 969 Splenocytes were isolated and stimulated with PMA, Ionomycin, and Brefeldin A. Subsequently, 970 splenocytes were surface stained for CD3, CD4, and CD8 followed by intracellular staining for 971 IFNγ (C,D) and IL-17A (E). Different groups of mice used are depicted in F. Statistical analysis 972 was performed using One-way ANOVA with Tukey's post-hoc test (A, C, D, E) or Two-way 973 ANOVA with Tukey's post-hoc test (B) to determine significance. Asterisks directly above bars 974 indicated significance compared to control group while asterisks with comparison bars denoted 975 significance between the indicated groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Two 976 biological replicates were performed, and data plotted. In vitro studies had 3 replicates. Statistical significance was determined using One-way ANOVA with Tukey's post-hoc test. (Fig 5A) . Serum was collected 42 days after the 2 nd vaccination as well as 1077 28 days post-infection. F1-V specific IgA was determined by ELISA. Titers were determined in 1078 triplicate. One-way ANOVA with Tukey's post hoc test was used to determine significant 1079 differences between groups. Asterisks indicate significance compared to naïve serum. **** 1080 p<0.0001.