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Paragraph: 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.

Question: What is an example  of the effect of immunity to the  delivery vector on the efficacy of vaccination?
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.