23.1 Introduction

Infection of ruminants, particularly cattle, by Mycobacterium avium subsp. paratuberculosis (MAP) is common in most countries and leads to sufficient clinical paratuberculosis to result in significant economic losses (Garcia and Shalloo, 2015; Kirkeby et al., 2016).

The principal reasons behind these failures remain far from clear but stem from the exqui­site ability of MAP to subvert immune responses allowing it to establish long-term intracellular persistence (Bastida and Juste, 2011; Frie et al., 2017). It is probable that factors inherent within the choice of strains, method of attenuation or vaccine delivery provide suboptimal induction of early innate and adaptive cellular mecha­nisms essential to clear MAP effectively during primary infection, especially in particularly susceptible animals such as neonates (Mortier et al., 2013).

The history of vaccine use (covered more fully in Chapter 22, this volume) includes the use of a variety of approaches including classi­cally derived, but poorly characterized, live at­tenuated or killed vaccine strains, MAP protein subunits and even MAP glycolipid extractions (Jolly et al., 2013). More recent studies reverted back to using live attenuated strains (LAV) but with improved definitions, derived by directed knockout of MAP genes involved in various as­pects of virulence. Early vaccine strains from France (Vallee et al., 1934) were probably not markedly attenuated (Doyle, 1964). They were used live (Stuart, 1965) and relied on subcu­taneous compartmentalization in an oil and pumice-based adjuvant to optimize antigen presentation, inhibit growth and prevent dis­semination (Wilesmith, 1982). Later, a combi­national preparation of live UK strains (316F, strain II and 2e) administered in an oil-based emulsion to sheep (Sigurdsson, 1960) and goats (Saxegaard and Fodstad, 1985) achieved some success but stocks were poorly maintained and are no longer available.

A single culture of 316F from the 1907s was used as an original seed stock for various live vaccine formulations tested in France and Hungary in the 1980s (Kormendy, 1994), in New Zealand up until 2002 (Begg and Griffin, 2005) and in a more contemporary study showed significant reduction in mortalities in Greek sheep over a 4-year period (Dimareli-Malli et al., 2013). Preliminary genomic studies of 316F and 316 v, a similar Weybridge strain used for MAP enzyme-linked immunosorbent assay (ELISA) testing in Australia from 1986 (Milner et al., 1987) suggested them to have a simi­lar gene complement to the virulent reference strain K-10. This was confirmed by microarray analysis, which also revealed that some subcul­ture stocks of 316F had gained large genomic deletions generated during long-term exposure in suboptimal minimal culture media used for maintenance (Bull et al., 2013).

Concerns about the use of live strains on the grounds of health and safety, short shelf life and potential spread to the environment led to the introduction of heat-killed, whole-cell vac­cines (Emery and Whittington, 2004). The only MAP vaccine licensed for use in the USA uses a killed strain of Mycobacterium avium subspe­cies avium (Rathnaiah et al., 2017). An indig­enous killed MAP strain isolated from bison has been used in some Indian studies (Singh et al., 2013); however, all other major formulations derive from a series of 316F strain subcultures grown in various types of liquid cultures and administered at different concentrations (Emery and Whittington, 2004) or as MAP cell wall­deficient preparations (Hines et al., 2007b). One commercial killed 316F vaccine (Gudair) has been trialled extensively and registered in a national control programme (Reddacliff et al., 2006).

A shift in direction beginning after 2005 towards development of subunit vaccines incor­porating immunodominant MAP proteins was initiated primarily to avoid the induction of sup­pressive immune responses by whole-cell vaccine components and circumvent problems of cross­reactions with tuberculin in cattle. The availabil­ity of genomic and proteomic (Leroy et al., 2007; Hughes et al., 2008) arrays provided tools iden­tifying a range of potential immunodominant targets. The breadth of immune stimulation, longevity and type of immunological memory provided by these vaccines is still poorly under­stood and, so far, despite testing with a variety of delivery systems and immune-enhancing sub­strates, very few subunit vaccines have shown significantly better reduction of faecal shedding than whole-cell killed adjuvanted vaccines and none has been taken sufficiently far to show protection from disease. The observations that live Bacillus Calmette-Guerin (BCG) can prime or generate ‘trained immunity' (Kleinnijenhuis et al., 2012) that can be enhanced through sub­sequent directed subunit boosting and protect against subsequent mycobacterial challenge

Table 23.1. Summary of live attenuated strain (LAV) candidates tested in animals.

382 T. Bull

MAP3694c-MAP3695

(MAPK10)

2E11 MAP_RS18945-MAP.

RS18950 JDIP316

Intragenic

Kanamycin

MDM₁ monocyte-derived macrophage;CFU. colony-forming unitsjp., Intraperitonealjv.. intravenous;sc. SubcutaneousTBS, Phosphatebuffered saline;LN, lymph node.

(Hawkridge et al., 2008; Kaveh et al., 2016) suggests live attenuated prime, subunit boost strategies may also be applicable for paratuber­culosis; however, this approach has not as yet been explored.

Stimulating the complex balance of immu­nological responses required to achieve effective immunity from any MAP vaccine over long peri­ods is particularly challenging. Humoral (Pooley et al., 2019) and mucosal (Weiss et al., 2006) im­munity may have some impact, but the priming and maintenance of appropriate Th1 responses is crucial (Coussens, 2004; Vordermeier and Hewinson, 2006). Vaccination approaches need optimizing against contributory factors includ­ing the possible requirement for boosting, type or breadth of antigens presented, differential responses to vaccination between neonates and adults (Koets and Grohn, 2015), and sensitiza­tion from encounters with other mycobacteria (Orme and Collins, 1985; Hope et al., 2005; Zimmermann et al., 2018). The mode of vac­cine delivery is also likely to provide a signifi­cant variable. Recent studies on Mycobacterium tuberculosis vaccination in non-human primates show that mucosal (Kaveh et al., 2016) or in­travenous vaccination (iv.) with whole-cell my­cobacterial vaccines provides better protection than parenteral subcutaneous (sc.), intradermal (id.) or intraperitoneal (ip.) vaccination, possibly elicited through improved induction of more T helper Th17 cells, resident memory T cells and effector T cells at these sites (Voss et al., 2018). As described in this chapter, the means of deliv­ery can be crucial and can affect efficacy. The optimal method however may be related to the type of vaccine being administered and this as yet remains an unknown.

The size of MAP reservoirs also suggests that exposure to MAP is almost inevitable and that even eradication programmes that include vaccination may not be successful unless the vac­cine used also has a therapeutic effect. Vaccines are aimed principally at prophylactic protection; however, there is evidence that selected delivery systems could be used therapeutically on in­fected animals (Bull et al., 2007; Santema et al.,

2013). The possibility that chronic MAP infec­tion in humans may be involved in the develop­ment of Crohn's disease raises the possibility that a vaccine could provide an alternative direction for treatment. A vaccine adapted for humans would need to act therapeutically and pass rigor­ous Phase I and Phase II safety trials, and one human vaccine has already begun development, with some success (Folegatti et al., 2019). The current goal in vaccine development however is firmly focused on providing better protection of ruminants against disease and elimination of shedding from subclinically infected animals. Many challenges lie ahead. Devising effective ways of designing, screening and testing vaccine candidates needs more work. Despite attempts to better standardize animal models (Hines et al., 2007a) there is little agreement on how vaccine testing in one species will predict protection in another (Bannantine et al., 2014a; Hines et al., 2014; Park and Yoo, 2016; Rathnaiah et al., 2017; Barkema et al., 2018).

Techniques for monitoring and predicting disease progression clearly remain suboptimal. This includes little knowledge of the relation­ship between faecal shedding and subclinical cattle disease (Corbett et al., 2018), low specific­ity with serum testing using MAP antigens and disease status (Bannantine et al., 2017), and the absence of a gold standard vaccine able to pro­vide sterilizing protection in any model. The lack of agreed correlates of protection able to screen for candidate vaccines prior to full challenge tri­als also remains a major roadblock to progress (Ganusov et al., 2015). Nevertheless, against this background of unknowns a few promising avenues of research have emerged, including two different prophylactic vaccines (Bull et al., 2014; Shippy et al., 2017) that have managed to abrogate MAP faecal shedding in cattle after high dose challenge, within a test environment.

23.2