Fish­ing With Al­gae For Malaria Vac­cines

by James Gre­gory

Malaria is a big killer and a ma­jor world­wide health con­cern. The num­ber of malaria-re­lated deaths has fallen to ap­prox­i­mately 650,000 in 2010, from well over 1 mil­lion just ten years ago, thanks to the World Health Or­ga­ni­za­tion (WHO) and to phil­an­thropic or­ga­ni­za­tions, in­clud­ing the Bill and Melinda Gates Foun­da­tion. This ef­fort, com­posed of drug-based treat­ment and in­sec­ti­cide-treated bed nets (ITNs), was noth­ing short of heroic, but un­for­tu­nately it is not sus­tain­able. Malaria strains have been re­sis­tant to chloro­quinones for decades and the emer­gence of artemisinin re­sis­tant strains is threat­en­ing the last ef­fec­tive drugs in our ar­se­nal click here and here. The story is sim­i­lar for in­sec­ti­cides. Coun­tries with in­sec­ti­cide-re­sis­tant mos­qui­tos far out­num­ber those with­out. For in­stance, mos­qui­tos in Cote D'Ivoire are re­sis­tant to four classes of in­sec­ti­cides, those in In­dia, to three.

Fig­ure 1. Malaria caus­ing Plas­mod­ium species un­dergo a com­plex three-stage life cy­cle. Each stage of the life cy­cle could po­ten­tially be tar­geted for vac­cine in­ter­ven­tion. Pre-ery­thro­cytic vac­cines – pro­tec­tion for the vac­ci­nated in­di­vid­ual (RTS,S is a pre-ery­thro- cytic vac­cine). Blood – re­duce the sever­ity of malaria in­fec­tions. Mos­quito – block malaria trans­mis­sion by pre­vent­ing mos­quito in­fec­tion. Source. Fron­tispiece: An al­gae farm, a po­ten­tial site of vac­cine pro­duc­tion. Source

In re­sponse, the Malaria Vac­cine Ini­tia­tive was es­tab­lished in 1999 by a grant from the Bill and Melinda Gates foun­da­tion to ac­cel­er­ate the de­vel­op­ment of ef­fec­tive malaria vac­cines. Vac­cines are the sin­gle most cost ef­fec­tive means of dis­ease pre­ven­tion, but, for malaria, com­ing up with one has not been easy. The most deadly form of hu­man malaria is caused by the pro­tist, Plas­mod­ium fal­ci­parum, which has about 5300 genes and three dis­tinct de­vel­op­men­tal stages each housed in a dif­fer­ent environment—liver, blood, and mos­quito (Fig. 1). P. fal­ci­parum spends the ma­jor­ity of its time hid­den in liver cells and red blood cells. Here it is se­questered from the anti­gen-pre­sent­ing cells that would trig­ger an adap­tive im­mune re­sponse. Fur­ther­more, P. fal­ci­parum is a mov­ing tar­get; its cell sur­face pro­teins are highly poly­mor­phic, likely a re­sponse to the se­lec­tive pres­sure im­posed by the im­mune sys­tem. It seems that in this arms race the par­a­site is win­ning, but there is hope.

Re­com­bi­nant pro­tein-based vac­cines, oth­er­wise known as sub­unit vac­cines, may be able to tip the bal­ance in our fa­vor. Dur­ing phase III clin­i­cal tri­als, a pre-ery­thro­cytic vac­cine based on sur­face pro­teins from the sporo­zoite (called RTS,S) pro­vided 30–50% pro­tec­tion over a 14 month pe­riod. While promis­ing, bet­ter per­for­mance is needed to move to­wards erad­i­ca­tion. Glax­o­SmithK­line has promised to sell RTS,S on the slimmest of mar­gins, but even so the price of such a vac­cine is of se­ri­ous con­cern given the num­ber and so­cioe­co­nomic sta­tus of peo­ple in malaria en­demic re­gions. Re­com­bi­nant pro­teins can be ex­pen­sive to pro­duce be­cause they must be pu­ri­fied from the ex­pres­sion sys­tem (in the case of RTS,S, from yeast). For­mu­la­tion with ad­ju­vants as well as is­sues with cold stor­age and in­jec­tion de­liv­ery also add to the cost and com­pli­cate the lo­gis­tics of wide­spread vac­ci­na­tion.

Fig­ure 2a. A C. rein­hardtii cell en­gi­neered to ex­press GFP, seen un­der a flu­o­res­cence mi­cro­scope. Source. May­field lab, UCSD.

An­other yeast-made sub­unit vac­cine had made it to clin­i­cal tri­als in 2008 via a dif­fer­ent strat­egy. This vac­cine tar­geted Plas­mod­ium sur­face pro­tein 25 (Pfs25). Dis­cov­ered by David Kaslow, now di­rec­tor of the Malaria Vac­cine Ini­tia­tive, Pfs25 is es­sen­tial for the sex­ual de­vel­op­ment of P. fal­ci­parum within the mos­quito. The idea was that an­ti­bod­ies to this pro­tein would be present in a vac­ci­nated in­di­vid­ual and would be taken up by the mos­quito dur­ing a blood meal. These an­ti­bod­ies would then pro­tect the mos­quito from in­fec­tion, thus pre­vent­ing sub­se­quent trans­mis­sion to a hu­man host. This so-called trans­mis­sion block­ing vac­cine is fea­si­ble be­cause hu­man malaria has no sig­nif­i­cant an­i­mal reser­voir and, un­like the plas­modial sur­face pro­teins made the liver and blood stages, this fam­ily of pro­teins is well-con­served. Keep in mind that block­ing malar­ial trans­mis­sion by elim­i­nat­ing the mos­quito vec­tor us­ing DDT was cru­cial to the suc­cess of the erad­i­ca­tion strate­gies in the United States and else­where.

Un­for­tu­nately, Phase I tri­als of the Pfs25 vac­cine were stopped due to an al­ler­gic re­ac­tion. The choice of yeast for Pfs25 pro­duc­tion may be partly to blame. How­ever, no suit­able al­ter­na­tives were avail­able at the time and pu­ri­fy­ing it from sex­ual stage P. fal­ci­parum par­a­sites is not fea­si­ble. Pfs25 and other mem­bers of this fam­ily are not gly­co­sy­lated, which is an im­por­tant dis­tinc­tion be­cause gly­co­sy­lated Pfs25 alone does not elicit trans­mis­sion block­ing an­ti­bod­ies. The gene en­cod­ing the Pfs25 sub­unit vac­cine used in yeast con­tained two mu­ta­tions to pre­vent N‑linked gly­co­sy­la­tion at two sites, but yeast is no­to­ri­ous for hy­per­gly­co­sy­lat­ing re­com­bi­nant pro­teins.

Fig­ure 2b. Func­tional char­ac­ter­i­za­tion of an­ti­bod­ies to al­gae-pro­duced Pfs25. Im­muno­flu­o­res­cence im­ages of sex­ual stage par­a­sites. Green – bound an­ti­bod­ies. Blue – DAPI stained DNA. Source

Bac­te­r­ial pro­duc­tion was un­suc­cess­ful, most likely be­cause Pfs25 has a com­plex ter­tiary struc­ture that con­tains tan­dem re­peats of epi­der­mal growth fac­tor (EGF)-like mo­tifs. EGF mo­tifs con­tain mul­ti­ple disul­fide bonds, but the cy­toso­lic re­duc­tion po­ten­tial in bac­te­ria in­hibits cys­teine-cys­teine bonds. This de­fi­ciency is of­ten over­come by shut­tling pro­teins to the periplasm, but E. coli-pro­duced Pfs25 did not elicit trans­mis­sion block­ing an­ti­bod­ies. Thus, the struc­ture was not suf­fi­ciently sim­i­lar to na­tive Pfs25.

Pro­duc­tion of Pfs25 pre­sented a rare op­por­tu­nity for the al­gae com­mu­nity to get in­volved be­cause of the unique bi­ol­ogy of the al­gal chloro­plasts. Chloro­plasts are prokary­otic in na­ture, de­scen­dants of a cyanobac­terium that was long ago en­gulfed by a eu­kary­otic cell and sub­se­quently lost most of its genes. Un­like their prokary­otic dis­tant rel­a­tives, such as E. coli, chloro­plasts can form disul­fide bonds, do not gly­co­sy­late pro­teins, and con­tain eu­kary­otic chap­er­ones. Fur­ther­more, al­gal pro­duc­tion is highly scal­able and eco­nom­i­cal when grown in out­door ponds. An en­tire in­dus­try is be­ing born on the premise that al­gae-based gaso­line can sup­plant the fi­nite oil re­serves, them­selves the re­sult of mil­lions of years' worth of fos­silized al­gae. Eighty-seven mil­lion bar­rels of oil are con­sumed daily on this planet—that's a lot of al­gae.

I have been in­volved in this al­gal work, and here is a brief ac­count of what we have achieved. We en­gi­neered the fresh­wa­ter alga, Chlamy­domonas rein­hardtii, to pro­duce Pfs25. Its chloro­plast can be trans­formed via par­ti­cle bom­bard­ment pro­vided the DNA-coated gold par­ti­cles are shot into the al­gae at ve­loc­i­ties high enough to pen­e­trate the cell wall, the outer cell mem­brane, and the dou­ble lay­ered chloro­plast mem­brane. Once the DNA-gold com­plex en­ters the chloro­plast, the aque­ous en­vi­ron­ment sol­u­bi­lizes the DNA, thereby re­leas­ing it from the gold. The DNA then in­te­grates into the chloro­plast genome by ho­mol­o­gous re­com­bi­na­tion. Much like bac­te­r­ial plas­mids, there are mul­ti­ple copies of the chloro­plast genome. Sev­eral rounds of se­lec­tion pro­motes re­com­bi­na­tion be­tween the mul­ti­ple copies of the genome in the sin­gle chloro­plast un­til even­tu­ally all of them con­tain the trans­gene.

The struc­ture of al­gae-pro­duced Pfs25 seems to re­sem­ble the na­tive pro­tein. Im­muno­flu­o­res­cence ex­per­i­ments us­ing an­ti­bod­ies to al­gae-pro­duced Pfs25 are con­sis­tent with the known lo­cal­iza­tion of Pfs25 (Fig. 2A) Pfs25 is an abun­dant ex­tra­cel­lu­lar pro­tein that is em­bed­ded in the outer mem­brane of sex­ual stage par­a­sites. Con­sis­tent with this, an­ti­bod­ies to al­gae-pro­duced Pfs25 ap­pear to se­lec­tively bind to ga­mete, ga­me­to­cyte, and zy­gote mem­branes. These data sug­gest that C. rein­hardtii may be a suit­able or­gan­ism for mak­ing Pfs pro­teins. Most grat­i­fy­ing is that these an­ti­bod­ies block malaria trans­mis­sion (Fig. 2B). This can be mea­sured in the lab­o­ra­tory us­ing a stan­dard mem­brane feed­ing as­say (SMFA). In this ex­per­i­ment, hu­man blood, in vitro cul­tured P. fal­ci­parum ga­me­to­cytes, and the mouse an­ti­bod­ies to al­gae-pro­duced Pfs25 are mixed to­gether in a small ap­pa­ra­tus that al­lows mos­qui­tos to feed through a mem­brane. This ex­per­i­ment sim­u­lates a mos­quito feed­ing on a per­son. The mos­qui­tos are dis­sected nine days later to look for the par­a­site. See­ing no par­a­sites in­di­cates trans­mis­sion block­ing ac­tiv­ity.

Fig­ure 2c. Stan­dard mem­brane feed­ing as­say. Mos­qui­tos in­fected with blood con­tain­ing an­ti­bod­ies to al­gae-pro­duced Pfs25 show a sig­nif­i­cant re­duc­tion in oocysts. Source

The ac­tiv­ity of an­ti­bod­ies raised to al­gae-pro­duced Pfs25 will have to be com­pared with other can­di­dates. Re­com­bi­nant Pfs25 made in to­bacco also elicited trans­mis­sion block­ing an­ti­bod­ies, and so did a vi­ral vec­tor vac­ci­na­tion strat­egy. How­ever, al­gae re­main the only sys­tem that does not re­quire mu­ta­tional al­ter­ation of the pro­tein to avoid gly­co­sy­la­tion, and it is likely the sys­tem best suited for large-scale, in­ex­pen­sive pro­duc­tion.
 

How­ever, these are all con­ven­tional vac­cines that re­quire a nurse and a nee­dle for ad­min­is­tra­tion.  How about an oral vac­cine? Malaria-en­demic coun­tries tend to be poor and to have un­der­de­vel­oped health in­fra­struc­tures, thus lack the re­sources to im­ple­ment dis­sem­i­na­tion of an in­jectable vac­cine. For these coun­tries, heat-sta­ble, orally-de­liv­ered vac­cines may be the an­swer. Al­gae and many other plants are deemed safe for con­sump­tion by the Food and Drug Ad­min­is­tra­tion. Oral vac­cines made in to­bacco and pota­toes in 1995 have largely been an un­re­al­ized dream (ex­cept for po­lio, but that's a dif­fer­ent story). In­deed, there are no com­mer­cially avail­able oral sub­unit vac­cines. Re­search labs are con­tin­u­ing to look for an­swers. Nearly 300 pa­pers re­lated to oral vac­cines have been pub­lished just this year (NCBI). Many of these stud­ies uti­lize anti­gens that are fused to or co-ad­min­is­tered with tox­ins from path­o­genic bac­te­ria, which ap­pear to mod­u­late the gut im­mune sys­tem and act as mu­cosal ad­ju­vants. So it seems even the 'bad' bac­te­ria are good some­times. I have only re­cently be­gun em­bark­ing in a ca­reer to­wards mak­ing vac­cines based on mod­ern tech­nol­ogy. I am hope­ful that ef­forts by my­self and oth­ers will bring re­sults that will help a lot of peo­ple in need.

Ref­er­ence

Gre­gory JA, Li F, To­mosada LM, Cox CJ, Topol AB, Vinetz JM, May­field S (2012). Al­gae-pro­duced pfs25 elic­its an­ti­bod­ies that in­hibit malaria trans­mis­sion. PloS one, 7 (5). PMID 22615931

Jamie is a post­doc in the May­field lab, San Diego Cen­ter for Al­gae Biotech­nol­ogy at the Uni­ver­sity of Cal­i­for­nia, San Diego.