key: cord-0753019-v812zn3r
authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
title: Pre-existing immunity against vaccine vectors – friend or foe?
date: 2013-01-03
journal: Microbiology
DOI: 10.1099/mic.0.049601-0
sha: f5bdf18567bb3760e1ce05008135f0270badbd5c
doc_id: 753019
cord_uid: v812zn3r

Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.

In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .

Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .

However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .

As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.

In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.

Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .

The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .

A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .

Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic 

Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.

Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.

It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .

Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.

Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.

Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.

Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .

There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .

As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.

Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .

Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .

All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .

For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.

In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.

To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity.

Humoral responses and salmonellosis protection in chickens given a vitamin-dependent Salmonella typhimurium mutant

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin

IMOJEV(1): a Yellow fever virus-based novel Japanese encephalitis vaccine

Recombinant Salmonella vaccines for biodefence

Oral delivery of foreign antigens by attenuated Salmonella: consequences of prior exposure to the vector strain

Expression of two Listeria monocytogenes antigens (P60 and LLO) in Lactococcus lactis and examination for use as live vaccine vectors

Prior immunologic experience potentiates the subsequent antibody response when Salmonella strains are used as vaccine carriers

Vectors to achieve selective expression of vaccine antigens within eukaryotic cells using Salmonella spp. as carrier strains

Cross-reactive cellular and humoral immune responses to Salmonella enterica serovars Typhimurium and Enteritidis are associated with protection to heterologous re-challenge

Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity

Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

The potential role of fowlpox virus in rational vaccine design

Existing antilisterial immunity does not inhibit the development of a Listeria monocytogenes-specific primary cytotoxic T-lymphocyte response

Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity

Listeria-based cancer vaccines that segregate immunogenicity from toxicity

Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebocontrolled, test-of-concept trial

Immunogenicity of high expression adenovirus-hepatitis B virus recombinant vaccines in dogs

Listeria monocytogenes. A model system for the molecular study of intracellular parasitism

Live oral Salmonella vaccines: potential use of attenuated strains as carriers of heterologous antigens to the immune system

Viruses as vaccine vectors for infectious diseases and cancer

Comparison of the abilities of different attenuated Salmonella typhimurium strains to elicit humoral immune responses against a heterologous antigen

Attenuated poxvirus vectors MVA and NYVAC as promising vaccine candidates against HIV/AIDS

A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag

Rapid protection of gnotobiotic pigs against experimental salmonellosis following induction of polymorphonuclear leukocytes by avirulent Salmonella enterica

Emergency and M. Saxena and others therapeutic vaccination -is stimulating innate immunity an option?

New recombinant Ad5 vector overcomes Ad5 immunity allowing for multiple safe, homologous immunizations

Impact of prior immunological exposure on vaccine delivery by Salmonella enterica serovar Typhimurium

Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization

Recombinant attenuated bacteria for the delivery of subunit vaccines

The poxvirus vectors MVA and NYVAC as gene delivery systems for vaccination against infectious diseases and cancer

Auxotrophic vaccines for tuberculosis

Induction of protective immunity to anthrax lethal toxin with a nonhuman primate adenovirus-based vaccine in the presence of preexisting anti-human adenovirus immunity

Expression of human immunodeficiency virus type 1 gp120 from herpes simplex virus type 1-derived amplicons results in potent, specific, and durable cellular and humoral immune responses

Immunogenicity of recombinant adenovirus-respiratory syncytial virus vaccines with adenovirus types 4, 5, and 7 vectors in dogs and a chimpanzee

Construction of highly attenuated Salmonella enterica serovar Typhimurium live vectors for delivering heterologous antigens by chromosomal integration

Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells

Recombinant Listeria monocytogenes as a live vaccine vehicle and a probe for studying cell-mediated immunity

Effect of attenuated Salmonella enterica serovar Typhimurium expressing a Streptococcus mutans antigen on secondary responses to the cloned protein

Humoral immune response induced by oral administration of S. typhimurium containing a DNA vaccine against porcine reproductive and respiratory syndrome virus

Attenuated Listeria monocytogenes vaccine vectors expressing influenza A nucleoprotein: preclinical evaluation and oral inoculation of volunteers

Potent immune responses and in vitro pro-inflammatory cytokine suppression by a novel adenovirus vaccine vector based on rare human serotype 28

Immunity to intracellular bacteria

Live attenuated bacterial vaccines

In vivo compartmentalization of functionally distinct, rapidly responsive antigen-specific T-cell populations in DNA-immunized or Salmonella enterica serovar Typhimurium-infected mice

Long-term immunological memory induced by recombinant oral Salmonella vaccine vectors

Effect of preexisting immunity to Salmonella on the immune response to recombinant Salmonella enterica serovar Typhimurium expressing a Porphyromonas gingivalis hemagglutinin

New insights on adenovirus as vaccine vectors

Reduced immune responses after vaccination with a recombinant herpes simplex virus type 1 vector in the presence of antiviral immunity

Impact of preexisting vector-specific immunity on vaccine potency: characterization of Listeria monocytogenes-specific humoral and cellular immunity in humans and modeling studies using recombinant vaccines in mice

Attenuated Salmonella typhi and Shigella as live oral vaccines and as live vectors

Vertical and horizontal transmission of Salmonella within integrated broiler production system

Viral vectors in malaria vaccine development

Magnitude and phenotype of cellular immune responses elicited by recombinant adenovirus vectors and heterologous primeboost regimens in rhesus monkeys

T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8 + T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules

Preexisting immunity to poliovirus does not impair the efficacy of recombinant poliovirus vaccine vectors

Salmonella: immune responses and vaccines

HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis

Induction of a homologous and heterologous invasion-inhibition effect after administration of Salmonella strains to newly hatched chicks

Impact of vector-priming on the immunogenicity of a live recombinant Salmonella enterica serovar Typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human volunteers

Equine viral vaccines: the past, present and future

Immune response to infection with Salmonella typhimurium in mice

Differential CD4 + versus CD8 + T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates

Role of CD4 + T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection

MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses

Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine

The induction of HIV Gag-specific CD8 + T cells in the spleen and gutassociated lymphoid tissue by parenteral or mucosal immunization with recombinant Listeria monocytogenes HIV Gag

Effect of preexisting immunity on an adenovirus vaccine vector: in vitro neutralization assays fail to predict inhibition by antiviral antibody in vivo

Pre-existing adenovirus immunity modifies a complex mixed Th1 and Th2 cytokine response to an Ad5/HIV-1 vaccine candidate in humans

Safety and immunogenicity of a replication-incompetent adenovirus type 5 HIV-1 clade B gag/pol/nef vaccine in healthy adults

Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon

Candidate influenza vaccines based on recombinant modified vaccinia virus Ankara

Prior immunity to homologous and heterologous Salmonella serotypes suppresses local and systemic anti-fragment C antibody responses and protection from tetanus toxin in mice immunized with Salmonella strains expressing fragment C

Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity

Viral vectors as vaccine platforms: deployment in sight

Influence of promoter, gene copy number, and preexisting immunity on humoral and cellular responses to a vectored antigen delivered by a Salmonella enterica vaccine

Newcastle disease virus: a promising vector for viral therapy, immune therapy, and gene therapy of cancer

Pre-existing anti-Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis

Heterologous prime-boost immunizations with different Salmonella serovars for enhanced antigen-specific CD8 T-cell induction

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity

Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity

Live attenuated Salmonella: a paradigm of mucosal vaccines

Listeria monocytogenes as a vaccine vector: virulence attenuation or existing antivector immunity does not diminish therapeutic efficacy

Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses

Pre-existing immunity to pathogenic Listeria monocytogenes does not prevent induction of immune responses to feline immunodeficiency virus by a novel recombinant Listeria monocytogenes vaccine

Immunogenicity and protective efficacy of a recombinant yellow fever vaccine against the murine malarial parasite Plasmodium yoelii

Strategies to overexpress enterotoxigenic Escherichia coli (ETEC) colonization factors for the construction of oral whole-cell inactivated ETEC vaccine candidates

Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae

Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together, on nontoxigenic E. coli bacteria

CD8 + -T-cell response to secreted and nonsecreted antigens delivered by recombinant Listeria monocytogenes during secondary infection

Production of adenovirus vectors and their use as a delivery system for influenza vaccines

Noncompetitive expansion of cytotoxic T lymphocytes specific for different antigens during bacterial infection

Vector priming reduces the immunogenicity of Salmonella-based vaccines in Nramp1 +/+ mice

Impact of vector priming on the immunogenicity of recombinant Salmonella vaccines

Immunogenicity of a Salmonella typhimurium aroA aroD vaccine expressing a nontoxic domain of Clostridium difficile toxin A

Avipoxviruses: infection biology and their use as vaccine vectors

Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria

Poxvirus-vectored vaccines for rabies-a review

Prior exposure to an attenuated Listeria vaccine does not reduce immunogenicity: pre-clinical assessment of the efficacy of a Listeria vaccine in the induction of immune responses against HIV

The immune response to a B-cell epitope delivered by Salmonella is enhanced by prior immunological experience

Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier

Pathogenicity and immunogenicity of a mutant strain of Listeria monocytogenes in the chicken infection model