弗雷迪·弗里希克特（Frischknecht）是海德堡大学传染病中心的教授。他获得了关于痘病毒如何移动并研究疟疾寄生虫的博士学位，已有15年了，主要专注于使用先进的成像技术来了解它们的移动方式。
Mirko Singer is a PhD student in the lab of Freddy Frischknecht. His work is focused on sporozoite surface molecules involved in motility and new ways to manipulate the parasite genome.
Can you explain how malaria vaccines work in general?
今年，第一种疟疾疫苗是正式的approved for use。但是，效率（预防严重疾病）是not very highand hence it’s large scale use is谁尚未推荐。This vaccine is based on a single protein and blocks the parasite before it enters the liver.
A single escaping parasite however will lead to a full infection. Other protein-based vaccines targeting blood stage antigens are awaiting or undergoing clinical trials. Considering the practically unlimited寄生虫在野外的分歧it is thus not surprising that a single protein vaccine does not provide complete protection.
Alternatives to protein-based vaccines are the use of liveattenuated parasites as vaccines。These are of course more complex to generate and deliver but can offer full sterile protection, albeit usuallyonly against the very same strain of parasite。
Originally whole mosquitoes with the parasites have been treated withγ-辐射。经过此治疗后，寄生虫仍然可以侵入肝脏，但无法在内部生长。这会导致细胞免疫反应和抗体产生，可以完全保护以下感染。
Similar effects can be achieved with the deletion of genes required for liver stage development, but so far, identification of targets that lead to a late arrest (resulting in increased immune responses) and arrest safely has been difficult. Another solution is the administration of untreated parasites and subsequent drug treatment.
What is the疟原虫生命周期，不同的疫苗在哪里适合？
疟原虫由蚊子传播。寄生虫被注入宿主的皮肤，在那里他们迁移以找到并进入血管。
然后，寄生虫感染了肝脏，该肝脏首次繁殖。这个阶段可以持续一周，没有症状。
The parasites then escape again into the blood where they cause malaria by repeatedly multiplying in red blood cells.
While only one parasite is enough to infect a human (or animal) there can be up to half a kilogram of parasites in the blood of an infected person.
Ultimately the parasites can be taken up by another feeding mosquito to further develop into new infective forms again.
大多数药物，包括Nobel prize winning artemisinin, target the parasite where it causes the disease, in the blood. Similarly, many vaccines are also aimed at preventing the infection in the blood. Yet, the first approved malaria vaccine is based on a protein that is found in the parasites that are transmitted by the mosquito. Antibodies against this protein can stop the parasite migration in the skin.
Current experimental vaccines are targeting three different stages: (i) before parasites enter the liver by blocking motility in the skin and hepatocyte invasion, (ii) parasite growth in the liver by a T-cell based response and (iii) the block of parasite invasion of the red blood cells.
The parasite provides a target for several minutes in the skin, more than a week in the liver but only seconds during red blood cell invasion. This, and the parasite numbers (around 5 – 100) for the first two and up to 1010for the parasites in the blood let many scientists favor those first two stages.
Another, so far underrepresented, approach is to also stop the parasite after the blood meal in the mosquito. This will be hard to apply though as it does not provide benefit to the immunized individual, but it could severely reduce global transmission.
What is the advantage of using genome editing-based techniques to fight疟原虫?
目前,基因编辑techniqu的主要用途es in malaria parasites is still basic research. Just like in other organisms, zinc finger nucleases (ZFNs) or CRISPR/Cas9 can be used to subtly change a gene of interest.
例如，已利用ZFN进行调查目前用于杀死寄生虫的药物的抗药性机制。能够遵循耐药性的出现和传播，例如反对阿耳马素。
到目前为止，我们几乎没有刮擦降低寄生虫的潜力。
就像在使用这些基因组编辑工具的任何其他生物中一样，它们也可以帮助理解这些寄生虫的基本生物学机制。虽然这种怪异的生物学通常会自身引人入胜，但差异可以用来寻找靶向寄生虫的新方法。因此，预计尤其是使用CRISPR/CAS9将帮助该领域进一步发展，因为它加快了遗传变化的寄生虫的生成。
到目前为止，我们几乎没有刮擦降低寄生虫的潜力。但是，使用基因组编辑来产生功能修饰的增益是无止境的，并为产生衰减的疫苗提供了许多选择。
如果将减毒的寄生虫用作疫苗，则需要完全减弱并完全安全。在这里，我们认为我们的寄生虫有潜力补充基因缺失的方法，以完全衰减而不损害免疫原性。
How do ZFNs work and why have you chosen to use these tools?
ZFNs are specifically designed DNA cutting enzymes that work in pairs. When each partner binds its target DNA sequence in the right distance and orientation to each other they come together and generated a double strand break between the two binding sites.
We designed our approach so that we could make use of a previously established ZFN pair. We postulated that a single induced break in the genome would lead to the loss of the part of the chromosome that is not anymore connected to the centrosome.
We integrated both ZFNs together with their target, a green fluorescent protein (GFP) reporter, into the middle of a parasite chromosome. We used promoters that are only active in the mosquito or liver stage part of the live cycle to drive ZFN expression, which allowed us to study the effect of the DNA double strand break on parasite viability.
在我们的实验模型中，我们很幸运能够有效地同源重组，而无需非同源末端连接（NHEJ）。这基本上消除了核酸酶将DNA整合到特定位点的需求（只要它不必不含标记）即可。这就是为什么我们想到ZFN的其他用途，而缺乏NHEJ非常方便。
To be able to efficiently use these tools to fight malaria, what do we have to do?
There will be at least two important aspects. One is the societal acceptance of genetically altered organisms, the other aspect are technological obstacles. Both are clearly formidable multi-step challenges.
从技术上讲，我们需要能够产生寄生虫在其生命周期中所需点的100％捕捉到100％的寄生虫。就我们的方法而言，这将是在肝脏阶段。在此阶段，大多数研究都是在啮齿动物模型系统中进行的，因此我们首先必须将这些发现转化为人类疟疾寄生虫。
我们将需要能够存储将用于疫苗接种的寄生虫。然后，我们需要能够在大多数疫苗传递时能够在肌肉内注入那些寄生虫。
There are five species of malaria parasites infecting humans of which two are a major public health threat but only one parasite species (恶性疟原虫) can be cultured.
此外，我们将需要能够存储将用于疫苗接种的寄生虫。然后，我们需要能够在大多数疫苗传递时能够在肌肉内注入那些寄生虫。理想情况下，一次疫苗接种后将获得全面保护。
Currently scientists use frozen parasites from irradiated mosquitoes that need to be在保护工作之前，至少五次注射静脉注射。And this protection is likely specific only to the very same strain, while there are potentially unlimited numbers of different strains infecting people.
In your study you found that ZFNs are not 100% efficient. Can you explain how the parasites managed to escape the ZFN-based attack?
After optimization, ZFNs were 100% effective in cutting the疟原虫基因组。然而我们发现时机expression was extremely important to get a developmental arrest of the parasites. If we expressed the ZFNs too late, some parasites could escape the liver without any genetic modification and initiate a blood stage infection.
If we expressed the ZFNs too early, the parasites were too sick to efficiently infect the liver. This is in a sense the equivalent of irradiating parasites too much or too little. We finally managed to express the ZFNs ‘just right’ but this indeed led in rare cases to an unexpected repair of the DNA break.
令我们惊讶的是，这些寄生虫可以在休息两侧的DNA中使用微论，以修复其染色体并生存，从而导致完全发育和疾病。即使很少发生这种情况，免疫所需的大量寄生虫通常会导致感染。
因此，我们必须将产生基于ZFN的缺失的策略与其他基因缺失策略相结合，以提高安全性。我们解释如何做到这一点in the discussion of the paper。We also highlight that it will be important to look not only at vaccine safety but also at efficacy. Despite much hype and hope we believe it is still rather early days for attenuate malaria vaccines.
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